Circulation of human coronaviruses OC43 and 229E in Córdoba, Argentina.

This is the first study of respiratory infections in Córdoba, Argentina, caused by endemic human coronavirus (HCoV)-OC43 and HCOV-229E, which circulated during 2011-2012 at a 3% rate, either as single or multiple infections. They were detected mainly in children, but HCoV-229E was also found in adults. HCoV-229E was detected in five out of 631 samples (0.8%), and HCoV-OC43 was found in 14 out of 631 (2.2%) samples. Clinical manifestations ranged from fever to respiratory distress, and a significant association of HCoV-229E with asthma was observed. Further studies and surveillance are needed to provide better clinical insights, early diagnosis, and medical care of patients, as well as to contribute to epidemiology modeling and prevention.

Comorbidity and high viral load linked to clinical presentation of respiratory human bocavirus infection.

Human bocavirus (HBoV) is a new parvovirus associated with acute respiratory tract infection (ARTI). In order to evaluate HBoV significance as an agent of acute respiratory disease, we screened 1,135 respiratory samples from children and adults with and without symptoms during two complete calendar years. HBoV1 prevalence in patients with ARTI was 6.33 % in 2011 and 11.64 % in 2012, including neonatal and adult patients. HBoV1 was also detected in 3.77 % of asymptomatic individuals. The co-detection rate was 78.1 %. Among children, 87 % were clinically diagnosed with lower respiratory infection (no significant differences between patients with and without coinfection), and 31 % exhibited comorbidities. Pediatric patients with comorbidities were significantly older than patients without comorbidities. Patients with ARTI had either high or low viral load, while controls had only low viral load, but there were no clinical differences between patients with high or low viral load. In conclusion, we present evidence of the pathogenic potential of HBoV1 in young children with ARTI. Since patients with HBoV1-single infection are not significantly different from those with coinfection with respect to clinical features, the virus can be as pathogenic by itself as other respiratory agents are. Furthermore, an association between high HBoV1 load and disease could not be demonstrated in this study, but all asymptomatic individuals had low viral loads. Also, children with comorbidities are susceptible to HBoV1 infection at older ages than previously healthy children. Thus, the clinical presentation of infection may occur depending on both viral load and the particular interaction between the HBoV1 and the host.

Members of Venezuelan Equine Encephalitis complex entry into host cells by clathrin-mediated endocytosis in a pH-dependent manner.

Pixuna virus (PIXV) and Río Negro virus (RNV) are mosquito-borne alphaviruses belonging to the Venezuelan Equine Encephalitis (VEE) complex, which includes pathogenic epizootic and enzootic subtypes responsible for life-threatening diseases in equines. Considering that the first steps in viral infection are crucial for the efficient production of new progeny, the aim of this study was to elucidate the early events of the replication cycle of these two viruses. To this end, we used chemical inhibitors and the expression of dominant-negative constructs to study the dependence of clathrin and endosomal pH on PIXV and RNV internalization mechanisms. We demonstrated that both viruses are internalized primarily via clathrin-mediated endocytosis, where the low pH in endosomes is crucial for viral replication. Contributing knowledge regarding the entry route of VEE complex members is important to understand the pathogenesis of these viruses and also to develop new antiviral strategies.

New insights into the antiviral activity of nordihydroguaiaretic acid: Inhibition of dengue virus serotype 1 replication.

BACKGROUND: Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4. PURPOSE: We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity. METHODS: The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes. RESULTS: NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process. CONCLUSION: The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process.

Human bocavirus 1 infection of CACO-2 cell line cultures.

Human bocavirus 1 (HBoV1) is a parvovirus associated with pneumonia in infants. It has been detected in different tissues, including colorectal tumors. In this study, we investigated whether Caco-2 cell line, derived from human colon cancer, can be utilized as a model for HBoV1 replication. We demonstrate HBoV1 replication in Caco-2 cultures supplemented with DEAE-dextran after inoculation with respiratory material from infected patients presenting with acute respiratory infection. A viral cycle of rapid development is displayed. However, in spite of HBoV1 DNA 4-fold increment in the supernatants and monolayers by day 1, evidencing that the system allows the virus genome replication after the entry occurred, infectious progeny particles were not produced. These results are consistent with an infection that is limited to a single growth cycle, which can be associated to mutations in the NS1 and VP1/VP2 regions of HBoV1 genome. Further research will contribute to fully elucidate these observations.

[First report of complete genome sequence and phylogenetic analysis of Human Bocavirus 1 isolated in Argentina]. / Primer reporte de secuencia genómica completa y análisis filogenético de Bocavirus Humano 1 aislado en Argentina.

ANTECEDENTS: Human bocavirus (HBoV) is a parvovirus identified for the first time in 2005 associated to upper- and lower- acute respiratory tract infection (ARI), which is one of the main causes of morbimortality in infant population worldwide. Currently four genotypes have been described named HBoV1-4, of which HBoV1 is the one predominantly related to ARI. OBJECTIVE: To obtain the complete genome of respiratory HBoV locally isolated. METHODS: By means of bioinformatics tools such as ClustalW and NCBI Primer-Blast, primers were designed to amplify overlapping DNA fragments altogether spanning the complete genome of HBoV. Fragments were amplified by PCR and sequenced by BigDye Terminator capillary technology. Sequence editing and phylogenetic analysis were accomplished using MEGA v6 software. RESULTS: Complete genome sequence of HBoV1 strain 307AR09 was obtained after isolation from respiratory secretion of a pediatric patient with bronchiolitis. The sequence was deposited in the GenBank public database (accession number KJ634207). The phylogenetic analysis including complete genome sequences of all four genotypes from around the world shows similarity close to 100% between the local strain and the virus originally discovered in Sweden (DQ000495). The four genotypes clustered in 2 groups of high internal homology: HBoV1-HBoV3 and HBoV2-HBoV4. CONCLUSIONS: We provide local molecular data that can be used in future technological developments for research and diagnostic tests intended for medical practice. Our results add support to the proposed redistribution of the four genotypes into 2 species.

Antecedentes. El Bocavirus humano (HBoV) es un parvovirus descripto por primera vez en 2005, asociado a cuadros leves y graves de infección respiratoria aguda (IRA), una de las principales causas de morbimortalidad en la población infantil en todo el mundo. Al presente se han identificado 4 genotipos, nombradas HBoV1 a 4, de los cuales el primero es el que se asocia a IRA con predominancia. Objetivo. Obtener el genoma completo de HBoV respiratorio aislado localmente. Métodos. Se diseñaron primers para fragmentos superpuestos del genoma completo de HBoV, empleando las herramientas informáticas ClustalW y NCBI Primer-Blast. Los fragmentos se amplificaron por PCR convencional y se secuenciaron mediante tecnología capilar BigDye Terminator. La edición de las secuencias y análisis filogenético se realizó con el programa MEGA v6. Resultados. Se obtuvo la secuencia genómica completa de HBoV1 cepa 307AR09, aislada de secreción respiratoria de paciente pediátrico con bronquiolitis. La misma fue depositada en la base de datos GenBank con número de acceso KJ634207. El análisis filogenético con secuencias genómicas completas de los 4 genotipos obtenidas en distintas regiones del mundo muestra similitud cercana al 100% con la secuencia original descubierta en Suecia (DQ000495), así como el agrupamiento de los 4 genotipos en 2 clusters de alta homología interna: HBoV1-HBoV3 y HBoV2-HBoV4. Conclusiones. Se aportan datos locales para futuros desarrollos tecnológicos destinados tanto a la investigación como al diseño de métodos diagnósticos para la práctica médica. Por otra parte, los resultados sustentan la propuesta de redistribución taxonómica de los 4 genotipos en 2 especies.

Human bocavirus respiratory infection in infants in Córdoba, Argentina.

UNLABELLED: It has been suggested that human bocavirus (HBoV) is related to acute respiratory infection (ARI) in children (prevalence: 0.9% to 33%) although clinical characteristics have not been clearly established yet. OBJECTIVES: To identify the presence of HBoV in patients with ARI hospitalized in Hospital de Niños de Córdoba and describe cases without co-infection. METHOD: HBoV screening was done by traditional PCR. Specimens to be screened were obtained from nasal secretions of 222 children under 2 years of age hospitalized due to an ARI during 2011. Demographic, clinical and radiological data were recorded. RESULTS: Fifteen HBoV+ patients (6.8%) were identified. Their median age was 3.5 months (range: 1-22), 7/15 in co-infection (5 respiratory syncytial virus, 1 parainfuenza-3, 1 Bordetella pertussis). Cases without co-infection: pneumonia 5/8, bronchiolitis 3/8; two required intermediate care, 7/8 oxygen therapy, 7/8 bronchodilators, 6/8 antibiotics; associated disease 1/8 (microcephalus/heart disease). CONCLUSIONS: HBoV was identified in 15 out of 222 children (6.8%); the diagnosis of pneumonia was predominant without severe cases nor complications upon discharge.

High frequency of human bocavirus 1 DNA in infants and adults with lower acute respiratory infection.

Human bocavirus (HBoV) is a parvovirus with a possible aetiological role in respiratory disease that is currently under investigation. We detected HBoV1 in children and adults hospitalized with acute disease of the lower respiratory tract. HBoV genome was detected by PCR in nasopharyngeal swabs collected from 75 patients aged 0-89 years during 2010. HBoV was found in 17/75 (22.7 %) patients, 64.7 % of them infants younger than 1 year old and 29.4 % adults older than 30 years [the bimodal age distribution among HBoV-positive (HBoV(+)) patients was statistically significant, P<0.001]. Of all HBoV(+) cases, 35.3 % were co-infected; all co-infections occurred in children (≤13 years old) and 83.3 % of them were HBoV-respiratory syncytial virus (RSV) co-infections. Among infants younger than 1 year, 50 % HBoV(+) specimens were co-infected, all of them with RSV. The rate of co-infection in infants was significantly higher compared to the frequency of co-infection in the whole cohort (P = 0.003). The results suggest that HBoV1 is involved in acute respiratory disease. Interplay between HBoV1 and RSV cannot be discarded as a cause of elevated percentages of co-detections in infants.

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

UNLABELLED: 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% 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% of the detections corresponding to infants less than 1 year old (among which the prevalence of HBoV was 26.3% in patients < 3 months of age, 22.1% in 3 to 6 months, 25.3% in 6 to 9 months, and 18.8% 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% 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.

Infección respiratoria por bocavirus humano en lactantes de Córdoba, Argentina / Human bocavirus respiratory infection in infants in Córdoba, Argentina

Bocavirus humano (BoVh) ha sido relacionado con la infección respiratoria aguda (IRA) en los niños (prevalencia 0,9% a 33%), aunque las características clínicas aún no han sido claramente establecidas. Objetivos. Identificar la presencia de BoVh en pacientes con IRA internados en el Hospital de Niños de Córdoba y describir los casos sin coinfección detectada. Método. Se realizó la pesquisa de BoVh por PCR convencional a partir de secreciones nasales de 222 niños menores de 2 años hospitalizados por IRA durante 2011 y se registraron los datos demográficos, clínicos y radiológicos. Resultados. Se identificaron 15 pacientes BoVh+ (6,8%), con una mediana de edad de 3,5 meses (rango 1 a 22), 7/15 en coinfección (5 virus respiratorio sincicial, 1 parainfluenza-3, 1 Bordetella pertussis). Casos sin coinfección: neumonía 5/8, bronquiolitis 3/8; dos requirieron cuidados intermedios, 7/8 oxigenoterapia, 7/8 broncodilatadores, 6/8 antibióticos; enfermedad asociada 1/8 (microcefalia/cardiopatía). Conclusiones. Se identificó BoVh en 15 de 222 niños (6,8%); predominó el diagnóstico de neumonía sin casos graves ni complicaciones al alta.

It has been suggested that human bocavirus (HBoV) is related to acute respiratory infection (ARI) in children (prevalence: 0.9% to 33%) although clinical characteristics have not been clearly established yet. Objectives. To identify the presence of HBoV in patients with ARI hospitalized in Hospital de Niños de Córdoba and describe cases without co-infection. Method. HBoV screening was done by traditional PCR. Specimens to be screened were obtained from nasal secretions of 222 children under 2 years of age hospitalized due to an ARI during 2011. Demographic, clinical and radiological data were recorded. Results. Fifteen HBoV+ patients (6.8%) were identified. Their median age was 3.5 months (range: 1-22), 7/15 in co-infection (5 respiratory syncytial virus, 1 parainfluenza-3, 1 Bordetella pertussis). Cases without co-infection: pneumonia 5/8, bronchiolitis 3/8; two required intermediate care, 7/8 oxygen therapy, 7/8 bronchodilators, 6/8 antibiotics; associated disease 1/8 (microcephalus/heart disease). Conclusions. HBoV was identified in 15 out of 222 children (6.8%); the diagnosis of pneumonia was predominant without severe cases nor complications upon discharge.

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.