Respuesta inmune y terapia gó©nica con vectores virales adenoasociados / Immune response and gene therapy with adenoassociated viral vectors

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

Human parvovirus B19 genotype 1 in suspected dengue patients of Tefé, Amazonas State, Brazil

Abstract INTRODUCTION: Human parvovirus B19 (B19V) is a common pathogen, which on infection causes variety of clinical conditions from benign self-limiting exanthematous disease and other similar pathologies to fetal death. METHODS: We collected 341 serum samples between the first and fourth day after the onset of symptoms from all patients suspected of dengue fever who were attended at Regional Hospital of Tefé. Initially, patients were screened for malaria by blood smear test and negative samples were sent to Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) situated in Manaus (AM) for dengue testing using semi-nested multiplex PCR. Further, we investigated 44 malaria and dengue-negative samples of children for B19V DNA by nested-PCR. Positive samples were analyzed by BLAST against entire public non-redundant nucleotide database and genotyped by phylogenetic analyses using neighbor-joining clustering method. RESULTS: Eight samples (18.2%) were found to be PCR positive. Fever, headache, ocular pain, and/or muscle pain were reported as the most frequent symptoms by the patients and none were diagnosed with rash at the time of sample collection. Phylogenetic analysis of major capsid protein 2 (VP2) and VP3 coding region showed high similarity with B19V genotype 1. CONCLUSIONS: Our results reveal the spread of B19V genotype 1 in Tefé. Moreover, our results emphasize the significance of laboratorial differential diagnosis using molecular techniques in patients with acute febrile, and thereby aid the health surveillance system in improving patient care even in the remote areas of Amazon.

Description of a human Bocavirus recombinant strain in the Americas

Human bocaviruses (HBoV) are mainly associated with respiratory and gastroenteric infections. These viruses belong to the family Parvoviridae, genus Bocaparvovirus and are classified in four subtypes (HBoV1-4). Recombination and point mutation have been described as basis of parvovirus evolution. In this study three viral sequences were obtained from positives HBoV sewage samples collected in two Uruguayan cities and were characterised by different methods as recombinant strains. This recombination event was localised in the 5' end of VP1 gene and the parental strains belonged to subtypes 3 and 4. These three Uruguayan strains are identical at the nucleotide sequences in the analysed genome region of the virus. As far as we known, this study represents the first detection of HBoV recombinants strains in the Americas.

Identification of co-infection by rotavirus and parvovirus in dogs with gastroenteritis in Mexico

ABSTRACT This is the first report on circulating canine rotavirus in Mexico. Fifty samples from dogs with gastroenteritis were analyzed used polymerase chain reaction and reverse transcription polymerase chain reaction in order to identify parvovirus and rotavirus, respectively; 7% of dogs were infected with rotavirus exclusively, while 14% were co-infected with both rotavirus and parvovirus; clinical signs in co-infected dogs were more severe.

Identification of Human Bocavirus type 4 in a child asymptomatic for respiratory tract infection and acute gastroenteritis - Goiânia, Goiás, Brazil

Abstract Human Bocavirus (HBoV) has been identified from feces and respiratory samples from cases of both acute gastroenteritis and respiratory illness as well as in asymptomatic individuals. The aim of this study was to detect and characterize HBoV from fecal samples collected from hospitalized children aged less than five years old with no symptoms of respiratory tract infection (RTI) or acute gastroenteritis (AGE). The study involved 119 children and one fecal sample was collected from each participant between 2014 and 2015. HBoV was detected using Nested-PCR, and the viral type identified by genomic sequencing. HBoV-4 was identified from one sample obtained from a hospitalized child with soft tissue tumor of the submandibular region. This is the first report of HBoV-4 identification in Brazil, but we consider that this type may be circulating in the country similar to the other types and new investigations are necessary.

Phylogenetic characterization of the first Ungulate tetraparvovirus 2 detected in pigs in Brazil

Abstract Ungulate tetraparvovirus 2 (UTV2) , formerly known as porcine hokovirus due to its discovery in Hong Kong, is closely related to a Primate tetraparvovirus (human PARV-4) and Ungulate tetraparvovirus 1 (bovine hokovirus). Until now, UTV2 was detected in European, Asian and North American countries, but its occurrence in Latin America is still unknown. This study describes the first report of UTV2 in Brazil, as well as its phylogenetic characterization. Tissue samples (lymph node, lung, liver, spleen and kidney) of 240 piglets from eight different herds (30 animals each herd) were processed for DNA extraction. UTV2 DNA was detected by PCR and the entire VP1/VP2 gene was sequenced for phylogenetic analysis. All pigs from this study displayed postweaning multisystemic wasting syndrome (PMWS). UTV2 was detected in 55.3% of the samples distributed in the variety of porcine tissues investigated, as well as detected in almost all herds, with one exception. The phylogenetic analysis demonstrated that Brazilian UTV2 sequences were more closely related to sequences from Europe and United States.

Bocavirus en niños menores de 5 años con infección respiratoria aguda: Provincia del Chaco, 2014 / Bocavirus in infants under 5 years with acute respiratory infection. Chaco Province, Argentina, 2014

La infección respiratoria aguda (IRA) es la patología más frecuente a lo largo de la vida de una persona y es la causa más común de morbi-motalidad en menores de 5 años. El objetivo de este trabajo fue determinar la frecuencia de bocavirus (BoV) en pacientes menores de 5 años con diagnóstico presuntivo de IRA en una ciudad capital del norte argentino (Resistencia, Chaco). Se analizaron aspirados nasofaríngeos correspondientes a 488 niños durante el año 2014. Los mismos fueron testeados por PCR en tiempo real hallándose BoV en 36 casos (7.4%), de los cuales 26 (72.2%) fueron infantes de 6-18 meses de vida. La mayor concentración de positivos se registró en el período junio-septiembre con un total de 28 pacientes (77.8%). Los casos positivos para BoV se observaron como infección única en el 50% de los mismos y el resto como infecciones concomitantes con otros microorganismos. No conocemos que haya otro estudio de epidemiología molecular de BoV en el norte argentino y destacamos la importancia de investigar los nuevos virus capaces de generar infección respiratoria aguda, y difundir el conocimiento de su circulación en la comunidad.

Acute respiratory infection (ARI) is the most frequent pathology along human life, being the most common cause of morbidity and mortality in children under 5 years. The aim of this study was to determine the frequency of bocavirus (BoV) in infants under 5 years with symptoms of ARI from north Argentina (Chaco province). The study was performed on nasopharyngeal aspirates from 488 patients, in the period of January-December 2014. The samples were tested by real time PCR and 36 positive BoV cases (7.4%) were detected. The period with the highest detection rate was June-September with 28 cases (77.8%), of which 26 (72.2%) were infants between 6-18 moths of life. In half of BoV positive cases this virus was detected as single infection of the upper respiratory tract, and in the remaining 50%, as concomitant infection with other microorganisms. To our knowledge, this would be the first study on molecular epidemiology of BoV in northern Argentina. We emphasize the importance of investigating these new viruses capable of generating acute respiratory disease and also to disseminate awareness on their circulation within the community.

Molecular and clinical evaluation of the acute human parvovirus B19 infection: comparison of two cases in children with sickle cell disease and discussion of the literature

Human parvovirus B19 is a well-known cause of severe conditions in patients with sickle cell disease, but the molecular mechanisms of the infection are insufficiently understood. The different clinical outcome of the acute parvovirus B19 infection in two pediatric patients with sickle cell disease has been examined. One of them developed life-threatening condition requiring emergency transfusions, while the other had asymptomatic infection, diagnosed occasionally. Both cases had high viral load and identical subgenotype, indicating that the viral molecular characteristics play a minimal role in the infection outcome.

Human bocavirus 1 and 3 infection in children with acute gastroenteritis in Brazil

To determine the positivity rate of human bocavirus (HBoV) 1 and 3 among children who presented with acute gastroenteritis symptoms during the period of 1994-2004 in the Central-West Region of Brazil, 762 faecal samples were tested using polymerase chain reaction (PCR) for the detection of HBoV DNA. Primers for a segment of the non-structural viral protein 1 (NS1) gene of HBoV-1 and HBoV-3 were used. Twelve HBoV-positive samples were further characterised via genomic sequencing and phylogenetic analysis. Of the samples tested, 5.8% (n = 44) were positive for HBoV-1 or HBoV-3 and co-infection was observed in 14 (31.8%) of the 44 HBoV-positive samples. Nine of the 14 samples were also positive for Rotavirus A and five were positive for Aichi virus. The genomic sequencing of the NS1 partial sequence of 12 HBoV-samples showed that 11 samples were characterised as HBoV-1 and that one was characterised as HBoV-3. The phylogenetic analysis showed that the HBoV-1 samples had a high sequence homology to others previously identified in China, Sweden and Brazil. This is the first study conducted in the Central-West Region of Brazil to detect HBoV-1 and HBoV-3 in faecal samples from children with acute gastroenteritis. Further studies are required to define the role of HBoVs as aetiological agents of gastroenteritis.

High prevalence of human bocavirus 1 in infants with lower acute respiratory tract disease in Argentina, 2007 - 2009

Human bocavirus (HBoV) is a parvovirus whose association with respiratory disease is currently under investigation. OBJECTIVE: To determine HBoV prevalence in children with lower acute respiratory infection. METHODS: We investigated HBoV in 433 nasopharyngeal aspirates collected in 2007-2009 from children 0 to 5 years old hospitalized with bronchiolitis or pneumonia in Córdoba, Argentina. RESULTS: The general prevalence of HBoV was 21.5 percent and the positive cases (HBoV+) were more frequent during winter and spring. The mean age of HBoV+ patients was 6.9 months, with 87.1 percent of the detections corresponding to infants less than 1 year old (among which the prevalence of HBoV was 26.3 percent in patients < 3 months of age, 22.1 percent in 3 to 6 months, 25.3 percent in 6 to 9 months, and 18.8 percent in 9 to 12 months). The sequence analysis of the NP1 coding region of 15 isolates showed that all isolates from Cordoba were HBoV1 which exhibited a homology of nearly 100 percent both among themselves and with the originally discovered virus from 2005. CONCLUSION: Overall, our results indicate that HBoV is a significant pathogen that contributes to acute respiratory infection both on its own and during coinfection with other viruses.

Bocavirus humano en Chile: características clínicas y epidemiológicas en niños con infecciones respiratorias / Human bocavirus in Chile: clinical characteristics and epidemiological profile in children with acute respiratory tract infections

Background: Human bocavirus (HBoV) is a newly discovered parvovirus found in children with acute respiratory tract infections (ARTI). Objectives: To describe the epidemiological and clinical profile of children < 5 years old consulting for ARTI, comparing cases of HBoV monoinfection and coinfection with other known respiratory viruses. Furthermore, we aimed to estimate the prevalence of viral shedding in asymptomatic children and perform phylogenetic analysis. Patients and Methods: We investigated the presence of HBoV in nasopharyngeal secretions from children consulting for AlRTI and among asymptomatic controls, between 2007 and 2008, by polymerase chain reaction. Results: HBoV was detected in 79 (21.8 percent) of 362 nasopharyngeal swabs obtained from children with ARTI. In 60/79 (76 percent), coinfection with other respiratory viruses was confirmed. Most common symptoms were cough, fever and rhinorrhea. Children infected only with HBoV showed significantly lower frequencies of respiratory distress, oxygen requirements and hospital admission than those with coinfection. HBoV was detected in 6/16 (37.5 percent) samples from asymptomatic children. The phylogenetic analysis of viruses from Chilean patients revealed that circulating HBoV was closely related to original strains. Conclusions: HBoV was found either in symptomatic and asymptomatic children. The severity of the disease was greater when HBoV was associated to other respiratory viruses.

Introducción: Bocavirus humano (HBoV) es un nuevo parvovirus encontrado en niños con infecciones respiratorias agudas (IRA). Objetivos: Describir la epidemiología y perfil clínico en niños < 5 años con IRA, comparando aquellos con HBoV como único agente identificado, con los que tenían co-infección con otro virus respiratorio. Además se evaluó su prevalencia en niños asintomáticos, y se realizó análisis filogenético. Materiales y Métodos: Se investigó la presencia de HBoV, por medio de reacción de polimerasa en cadena, en muestras de secreción nasofaríngea obtenida en niños con IRA y en controles asintomáticos, entre 2007 y 2008. Resultados: Se detectó HBoV en 79 (21,8 por ciento) de 362 muestras obtenidas en pacientes con IRA. En 60/79 (76 por ciento), se demostró co-infección. Los síntomas más frecuentes fueron tos, fiebre y rinorrea. Los pacientes con HBoV como único agente identificado mostraron frecuencias significativamente menores de dificultad respiratoria, requerimiento de oxígeno y hospitalización, comparado con los co-infectados. HBoV se detectó en 6/16 (37,5 por ciento) muestras de niños asintomáticos. El análisis filogenético de las cepas chilenas demuestra estrecha relación con las cepas originales. Conclusiones: HBoV está presente en niños chilenos con IRA y asintomáticos. La gravedad de la enfermedad fue mayor en el grupo con co-infección.

Bocavirus humano: Estudios en la literatura médica y en Chile / Human bocavirus: Studies in the literature and in Chile

The human bocavirus (HBoV), virus of the Parvoviridae family, discovered by molecular methods in 2005,has been reported in respiratory samples, stool, urine and blood, both in children and adults. Prevalence ratesrange from 0.8% in fecal samples of individuals with acute diarrhea, up to 19% in respiratory samples and blood.HBoV has been detected in up to 43% of nasopharyngeal samples in asymptomatic children. In Chile, HBoV wasdetected in 24.2% of nasopharyngeal swabs in children under 5 years of age with respiratory symptoms of which74% had coinfection with other viruses. In asymptomatic children under 5 years of age, 37.5% of NP sampleswere positive for HBoV. We discuss the role of HBoV as a causal agent of respiratory and/or enteric disease inlight of the high rates of coinfection and asymptomatic infections.

Seroepidemiological study of human parvovirus B19 among human immunodeficiency virus-infected patients in a medium-sized city in Rio de Janeiro, Brazil

Parvovirus B19 (B-19) may cause chronic anaemia in immunosuppressed patients, including those infected with human immunodeficiency virus (HIV). We studied single serum samples from 261 consecutive HIV-infected patients using an enzyme immunoassay to detect IgG antibodies to B-19. The seroprevalence of B-19-IgG was 62.8 percent. The differences in seroprevalence across gender, age, educational categories, year of collection of the serum samples, clinical and antiretroviral therapy characteristics, CD4+ count, CD4+ and CD8+ percentage and CD4+/CD8+ ratios were neither substantial nor statistically significant. There was a non-significant, inverse association between B-19 seropositivity and plasma HIV load and haemoglobin level. Our results indicated that 37.1 percent of patients might be susceptible to B-19 infection and remained at risk for being infected, mainly during epidemic periods. As B-19 infection can be treated with immune globulin preparations, it may be included in the diagnostic approach toward chronic anaemia in HIV-infected patients.

Molecular basis for porcine parvovirus detection in dead fetuses

Reproductive failures are still common grounds for complaint by commercial swine producers. Porcine parvovirus (PPV) is associated with different clinical reproductive signs. The aim of the present study was to investigate PPV fetal infection at swine farms having ongoing reproductive performance problems. The presence of virus in fetal tissues was determined by nested-polymerase chain reaction assay directed to the conserved NS1 gene of PPV in aborted fetuses, mummies and stillborns. Fetuses show a high frequency of PPV infection (96.4%; n = 28). In 60.7% of the fetuses, PPV were detected in all tissue samples (lung, heart, thymus, kidney, and spleen). Viral infection differed among fetal tissues, with a higher frequency in the lung and heart (p < 0.05). Fetuses with up to 99 days of gestational age and from younger sows showed a higher frequency of PPV (p < 0.05). No significant difference in the presence of PPV was detected among the three clinical presentations. The results suggest that PPV remains an important pathogenic agent associated with porcine fetal death.

The investigation of parvovirus B19 infection in patients with haematological disorders by using PCR and ELISA techniques

Parvovirus B19 has a marked tropism for erythroid progenitor cells. This may lead to chronic anemia in predisposed individuals. The purpose of the study was to investigate the frequency of parvovirus B19 infections in patients with diagnosis of haematological disorders. In order to determine the diagnostic use of different markers of parvovirus B19 infection, serum specimens obtained from 79 patients with haematological disorders were tested for specific antibodies and viral DNA through the use of ELISA and PCR techniques. Evidence of parvovirus B19 infection was found in 23/79 (29.1 percent) patients by demonstrating viral DNA and/or specific IgM antibody. B19 infection was established in 3 of 11 patients with chronic myeloid leukemia, in 3 of 11 acute myeloid leukemia, in 2 of 11 patients with multiple myeloma, in 3 of 8 patients with Hodgkin's lymphoma, in 5 of 10 patients with non-Hodgkin's lymphoma, in 1 of 6 patients with myelodysplastic syndrome, in 4 of 11 patients with chronic lymphocytic leukemia, and in 2 of 11 patients with acute lymphocytic leukemia. In 4 of 23 positive patients, only parvovirus B19 DNA could be detected, while 7 patients were tested positive for both parvovirus B19 DNA and specific IgM. Nine patients were tested positive for both B19 DNA and specific IgG. In the remaining 3 positive patients only specific IgM could be detected. Due to the discrepancies between DNA and IgM results, the diagnostic procedures should include a search for specific DNA by PCR methods if specific IgM has been found to be negative.

Erythrovirus B19 infection in acquired immunodeficiency syndrome: screening by histopathology, immunohistochemistry, and in situ hybridization

Erythrovirus B19 infects erythrocytic progenitors, transiently interrupting erythropoiesis. In AIDS patients it causes chronic anemia amenable to treatment. We looked for evidences of B19 infection in stored bone marrow material from patients with acquired immunodeficiency syndrome. Histological sections were made from stored paraffin blocks from 33 autopsies (39 blocks) and 35 biopsies (45 blocks, 30 patients) performed from 1988 to 2002. They were examined after hematoxylin-eosin (HE) staining, immunohistochemical (IHC), and in situ hybridization. HE revealed intra-nuclear inclusion bodies ("lantern cells") suggesting B19 infection in 19 sections corresponding to 19 of 63 patients examined with this test. Seven of 78 sections subjected to immunohistochemistry were positive, corresponding to 7 of 58 patients examined with this test. Fourteen sections corresponding to 13 of the 20 HE and/or IHC positive patients were subjected to in situ hybridization, with six positives results. Among the 13 patients subjected to the three techniques, only one gave unequivocal positive results in all and was considered a true positive. The frequency of B19 infection (1/63 patients) in the material examined can be deemed low.

Detection of human parvovirus B19 nonstrutural protein DNA by nested-polymerase chain reaction in gravida serum and pregnant tissues / 华中科技大学学报（医学）（英德文版）

A new nested-polymerase chain reaction (nested-PCR) assay was developed to detect human parvovirus B19 DNA corresponding to the nonstructural protein in clinical specimens in a routine diagnostic laboratory. The sensitivity of this highly specific assay was up to 0.005 fg of B19 DNA. Parvovirus B19 was identified in sera of 20 pregnant women with abnormal pregnant outcome. Among these 20 cases, intrauterine parvovirus infection did exist in 7 pregnant women because parvovirus B19 DNA was detected in the pregnant tissues of them such as placenta tissues, chorionic villi, amniotic fluid, fetal spleen, liver and abdominal fluids.

Parvovirus B19 infections in state of Rio de Janeiro, Brasil: 526 sera analyzed by IgM-enzyme-linked immunosorbent assay and polymerase chain reaction

In this study were analyzed 526 sera; the patients aged from two days to 65 years old presenting exanthema, which was the most frequent symptom observed, besides fever, adenomegaly, and arthralgia. These sera were negative by enzyme-linked immunosorbent assay (IgM-ELISA) for either rubella (495), toxoplasma (41), cytomegalovirus (12), measles (40), dengue (56), and they were submitted to nested polymerase chain reaction (PCR) for B19 DNA and commercial IgM-ELISA for B19. In 39 abortion cases, IgM or DNA were not detected, therefore they were not took into account for analysis. Specific DNA and IgM were detected respectively in 71 (14.5 percent) and IgM in 62 (12.7 percent) sera from 487 sera analyzed. IgM and DNA were simultaneously detected in 43 (8.8 percent), while agreement among the results by PCR and IgM-ELISA was observed in 440 (90.4 percent). The sera were collected from January 1999 to December 2000, most of them in 1999 (325), during winter and spring. The major number of clinical cases was observed in the age group from one to ten years old. IgM or DNA were detected in 23 from 51 municipal districts of the state of Rio de Janeiro, where the samples were collected.

Diagnóstico virológico de un brote de fiebre y rash producido por Parvovirus B19, Cuba, 1995 / Virologycal diagnosis of an aoutbreak of fever and rash caused by Parvovirus B19, Cuba, 1995

Se reportan los resultados obtenidos en el estudio de un brote de fiebre y rash ocurrido en Ciudad de La Habana en marzo de 1995. En las muestras de 35 pacientes se descartaron dengue, sarampión, rubéola, herpes simple y Epstein Barr como agentes causales del brote. Mediante la detección de anticuerpos IgM y la técnica de reacción en cadena de la polimerasa (RCP) se identificó al Parvovirus B19 como agente causal del brote. En 14/18 muestras (77,7 por ciento) se comprobó la infección por este agente por alguna de las técnicas empleadas. Este estudio se refiere al primer brote confirmado de Parvovirus B19 en Cuba