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BACKGROUND Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS Our findings provide evidence for the origin and spread of deletion variants and emphasise indels’ importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.

https://doi.org/10.1590/0074-02760210275

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state
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Hochmann, Jimena

; Parietti, Felipe

; Martínez, Jennyfer

; Lopez, Ana C

; Carreño, Mara

; Quijano, Celia

; Boccardo, Enrique

; Sichero, Laura

; Möller, Matías N

; Mirazo, Santiago

; Arbiza, Juan

Memórias do Instituto Oswaldo Cruz

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2020, Volume 115 elocation e190405

Abstract: En | Text: En | PDF: En

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

https://doi.org/10.1590/0074-02760190405 1303 downloads

CELL CULTURE ISOLATION OF HEPATITIS E VIRUS GENOTYPE 3 STRAIN OBTAINED FROM HUMAN FECES
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QUINTERO-GIL, Carolina

; MIRAZO, Santiago

; PARRA-SUESCÚN, Jaime

; LÓPEZ-HERRERA, Albeiro

; MAINARDI, Victoria

; ARBIZA, Juan

; ORDÚZ, Sergio

Acta Biológica Colombiana

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dec 2019, Volume 24 N. 3 Pages 503 - 508

Abstract: Es En | Text: Es En | PDF: Es | PDF: En

RESUMEN El virus de la hepatitis E (HEV) se considera como una de las principales causas de hepatitis viral aguda en el mundo; cada año ocurren aproximadamente 20 millones de infecciones y 57 000 muertes. Debido a la ausencia de un modelo de cultivo celular consenso, se sabe poco sobre el ciclo replicativo del virus. La línea celular A549 se considera susceptible al genotipo 3 de HEV, pero tanto la cepa viral como las condiciones del cultivo celular podrían afectar el aislamiento viral in vitro. Por tanto nos propusimos aislar in vitro una cepa genotipo 3 del HEV. Para ello, se inocularon células A549 con una cepa HEV-3 identificada previamente por caracterización genética, y se incubó durante dos horas a 37 °C. Cinco días después de la infección, las células se pasaron (subcultivaron) por primera vez, y se realizaron pases seriados cada cuatro días en promedio, durante 41 días. En cada pase se evalúo la replicación del HEV mediante RT-qPCR. La reinfección de la línea celular con progenie viral derivada de monocapas de A549 infectadas se evaluó mediante inmunofluorescencia y RT-qPCR. Se detectó ARN viral en cada pase a partir de monocapas, y el pico máximo se alcanzó a los 26 días post infección (2 x 106 copias/µL). En el ensayo de reinfección, se detectó antígeno de cápside perinuclearmente y formando focos, y se detectaron 1 x 104 copias/µL de RNA viral a las 96 horas post infección. El HEV recuperado de lisado de monocapas fue infeccioso. Este aislado viral ofrece una herramienta importante para estudiar aspectos desconocidos de la infección por HEV.

ABSTRACT Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/µL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/µL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.

https://doi.org/10.15446/abc.v24n3.79351 459 downloads

Evaluation of four viroid RNA extraction methods for the molecular diagnosis of CEVd in Citrus lemon using RT-PCR, Dot blot and Northern blot
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Umaña, Rodolfo

; Pritsch, Clara

; Arbiza, Juan R

; Rivas, Fernando

; Pagliano, Gabriela

Biotecnología Aplicada

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jun 2013, Volume 30 N. 2 Pages 131 - 136

Abstract: En Es | Text: En Es | PDF: En | PDF: Es

An efficient method for RNA extraction that leads to RNA high yield and purity is a technical issue relevant for development and optimization of molecular diagnostic methods aimed to detect viroid infections in citrus varieties. Residual contaminants may affect RNA detection depending on the molecular diagnosis approaches. This condition can be evaluated through RNA absorption spectrum analysis. Functionally, it is assessed through observation of RT-PCR amplification products and Northern blot and Dot-blot signal intensities, displaying levels of analytical response/sensitivity. Four RNA extraction methods were evaluated to determine their effects on the capacity to detect viroid CEVd presence/absence in Citrus limon through four molecular diagnostic approaches: 1) conventional viroid extraction (CVE); 2) phenol/guanidine thiocyanate (PGT), 3) SDS/potassium acetate (SPA); and 4) formaldehyde/ SSC (FS). Phloem tissue quantifications showed values between 7500 ng/µL and 1200 ng/µL and ranged 1.3-2.0 OD260/280. Evaluations through RT-PCR showed the expected amplifications of the entire CEVd genome, but erratic scenarios still remained. Non-radioactive probe hybridization techniques revealed high intensity signals (132 RU) for infected tissue, by using the CVE method, and a positivity cut-off for the presence of infection was established (78 RU). Nevertheless, molecular hybridization tools can jeopardize the diagnosis due to the thoroughness of the protocol and the RNA template conditions. The diagnostic ability of the association of Northern blot with CVE viroid extraction analyses as starting point was evidenced for successful detection, among the molecular methods tested.

En el desarrollo de metodologías moleculares diagnósticas en variedades cítricas propensas a infección viroide, se precisa la extracción eficiente de ARN, siguiendo criterios de concentración y pureza. Los contaminantes remanentes pueden afectar la detección según la herramienta molecular escogida. Esta condición se analiza a partir del espectro de absorción del ARN; mediante la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) y por los niveles de intensidad de las señales de Northern blot y Dot blot, en términos de respuesta analítica/sensibilidad. Se evaluaron cuatro métodos de extracción de ARN, por sus efectos sobre la detección de presencia/ausencia del Citrus Exocortis Viroid (CEVd) en Citrus limon mediante análisis moleculares diagnósticos: 1) extracción viroide convencional (EVC); 2) fenol/tiocianato de guanidina (FTG); 3) SDS/acetato de potasio (SAP); y 4) formaldehído/SSC (FS). Los valores del tejido de floema estuvieron entre 7500 ng/µL y 1200 ng/µL y los rangos entre 1.3 y 2.

121 downloads

Isolation and characterization of canine parvovirus type 2c circulating in Uruguay
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Pintos, Andrea Blanc

; Larrama, Cecilia Beatriz Negro

; Baratta, Eduardo Enrique Reolon

; Barthe, Mabel Beatriz Berois

; Rodonz, Juan Ramón Arbiza

Ciência Rural

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aug 2011, Volume 41 N. 8 Pages 1436 - 1440

Abstract: Pt En | Text: Pt En | PDF: Pt | PDF: En

Este trabalho relata o primeiro isolamento do CPV-2c em cultura de células (linhagem celular de fibroma canino A-72), no Uruguai. Os isolados foram obtidos a partir de 13 suabes retais de cães uruguaios com parvovirose. As amostras foram submetidas à reação em cadeia da polimerase (PCR) com dois pares de primers, polimorfismo de comprimento de fragmentos de restrição (RFLP), sequenciamento parcial do gene que codifica a proteína capsidial VP2 e caracterização filogenética. A cepa isolada foi confirmada como CPV-2c. Os resultados contribuem para um melhor conhecimento das cepas do CPV circulantes no Uruguai e incitam uma maior investigação sobre a eficácia das vacinas produzidas com cepas heterólogas utilizadas atualmente para proteger contra cepas circulantes.

This research reports the first CPV-2c isolation in cell culture (canine fibroma cell line A-72) in Uruguay. The isolates were obtained from 13 rectal swabs of Uruguayan dogs with parvovirosis. Samples were submitted to PCR with two sets of primers, restriction fragment length polymorphism (RFLP), partial sequencing of the gene encoding for VP2 capsid protein and phylogenetic characterization. The strain isolated was confirmed as CPV-2c. These results contribute to a better knowledge of CPV strains circulating in Uruguay and promote an evaluation of the efficacy of heterologous vaccines used to protect against the circulating strains.

5057 downloads

Investigación de Metapneumovirus humano en pacientes hospitalizados: estudio multicéntrico
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Medici, Conrado

; Crapuchetti, Cecilia

; Mattiauda, Alejandra

; Frabasile, Sandra

; Pizzorno, Andrés

; Masner, Martín

; Machin, Cecilia

; Silveira, Rosanna

; Zeballos, Jimena

; Galazka, Yeannette

; Batthyany, Lara

; Estevan, Miguel

; Arbiza, Juan

; Rubio, Ivonne

; Saráchaga, Ma Julia

Archivos de Pediatría del Uruguay

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dec 2010, Volume 81 N. 4 Pages 231 - 238

Abstract: Es En | Text: Es En | PDF: Es | PDF: En

Introducción: las infecciones respiratorias agudas bajas (IRAB) son la primer causa de hospitalización a lo largo del año. La etiología viral es la más frecuente. El Metapneumovirus humano (MNVh) ha sido vinculado a las IRAB con aspirado negativo para Virus respiratorio sincicial (VRS) y Adenovirus (AD). Objetivo: determinar la prevalencia, epidemiología, clínica y severidad de las infecciones por MNVh, e intentar detectar un patrón radiológico relacionado con el mismo. Métodos: se estudiaron niños entre 0 y 2 años de edad internados por IRAB en el Centro Hospitalario Pereira Rossell, Hospital Central de las Fuerzas Armadas, Hospital Policial, Hospital Británico y Asociación Española Primera de Socorros Mutuos en el período 1 de abril al 30 de noviembre de 2006. Los aspirados nasofaríngeos fueron analizados en la Sección Virología de la Facultad de Ciencias. Resultados: se estudiaron 185 pacientes, obteniéndose 17 resultados positivos para MNVh (9,2%), con 9 coinfecciones con VRS. La mayoría de los pacientes tenían 6 meses o menos de edad. Las manifestaciones clínicas principales fueron polipnea, tirajes y sibilancias. Los principales hallazgos radiológicos fueron infiltrado intersticial difuso e hiperinsuflación. Ningún paciente requirió internación en unidad de cuidados intensivos y no hubo casos fatales. Conclusiones: la prevalencia, características clínicas y evolutivas de las infecciones por MNVh no mostraron diferencias frente a las producidas por el VRS.

Introduction: acute lower respiratory tract infections (ALRTI) are the first cause of hospitalization throughout the year, increasing during winter months. Viral infections are the most frequent etiology. Human metapneumovirus (hMPV) has been linked to lower respiratory tract infections with a negative nasopharyngeal aspirate for Respiratory sincytyal virus (RSV) and Adenovirus (AD). Objectives: establish the prevalence, epidemiology, clinical presentation and severity of hMPV infections, and try to determinate a radiologic pattern related to this virus. Methods: we studied infants between 0 to 2 years of age hospitalized with ALRTI at the Centro Hospitalario Pereira Rossell, Hospital Central de las Fuerzas Armadas, Hospital Policial, Hospital Británico and Asociación Española Primera de Socorros Mutuos during the period 01/04/06-30/11/06. The nasopharyngeal aspirates were analyzed at Sección Virología of the Facultad de Ciencias. Results: out of the 185 samples analyzed, 17 hMPV positive cases (9.2%) were found, with 9 hMPV-RSV coinfections. Most patients were 6 months of age or less. The main clinical manifestations were tachypnea, chest indrawings and wheezing. Hyperinsuflation and diffuse interstitial infiltrates were the most frequent radiologic findings. None of the patients required admission at intensive care units and there were no fatal cases. Conclusions: the prevalence, clinical presentation and evolution of hMPV infections didn´t show differences with RSV infections.

3515 downloads Cited 1 time in SciELO

Meningitis y encefalitis víricas en Uruguay: Relevamiento mediante técnicas de reacción en cadena de polimerasa aplicadas al líquido cefalorraquídeo de los grupos herpes, enterovirus y arbovirus como principales agentes etiológicos. A propósito de 59 casos
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Salamano, Ronald

; Scavone, Cristina

; Baz, Mariana

; Rey, Andrea

; González, Gabriel

; Perna, Abayubá

; Cardinal, Pablo

; Lewin, Sara

; Arbiza, Juan

; Ruchanski, Dora

Revista Médica del Uruguay

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dec 2009, Volume 25 N. 4 Pages 212 - 218

Resumen Introducción: en nuestro país no existen trabajos sistemáticos relativos a la incidencia de virus que provoquen encefalitis y meningitis. Sí existen trabajos realizados en las décadas de 1960 y 1970 sobre seroprevalencia de arbovirus y poliovirus. Mediante la técnica de reacción en cadena de polimerasa (PCR) aplicada al líquido cefalorraquídeo (LCR) hoy es posible realizar en un breve lapso de tiempo un diagnóstico de certeza sobre diversos agentes virales responsables de estas neurovirosis. Material y métodos: se exploró la incidencia de virus de la familia herpes, enterovirus y grupo arbovirus mediante técnicas de PCR aplicadas al LCR en pacientes VIH negativos. Resultados: este trabajo presenta a 59 pacientes VIH negativos que padecieron encefalitis y meningitis de presumible etiología viral. Estos agentes son los responsables de la mayor cantidad de meningitis y encefalitis que suceden en nuestro continente. Conclusiones: el diagnóstico virológico final es posible realizarlo en más de la mitad de los casos presentados, predominando virus de la familia herpes tanto en niños como en adultos, no siendo despreciable la incidencia de enterovirus. No se detectó en este trabajo la presencia de arbovirus.

Résumé Introduction: dans notre pays, il n'existe pas de travaux systématiques relatifs à l'incidence des virus qui provoquent encéphalite et méningite. Il existe des travaux sur séroprévalence d'arbovirus et poliovirus faits entre 1960 et 1970. Au moyen de la technique d'amplification en chaîne de polymérase (PCR) appliquée au liquide céphalora-chidien (LCR), il est aujourd'hui possible de réaliser promptement un diagnostic certain sur divers agents viraux responsables de ces neuroviroses. Matériel et méthode: on explore l'incidence de virus de la famille herpès, entérovirus et arbovirus par des techniques de PCR appliquées au LCR chez des patients VIH négatifs. Résultats: ce travail présente 59 patients VIH négatifs qui subissent encéphalite et méningite de probable étiologie virale. Ces agents sont les responsables de la plupart des méningites et des encéphalites présentes dans notre continent. Conclusions: le diagnostic virologique final est possible pour la moitié des cas présentés, étant le virus de famille herpès prédominant chez les enfants et les adultes, l'incidence d'entérovirus restant importante. La présence d'arbovirus ne fut pas repérée.

Resumo Introdução: no nosso país não existem estudos sistemáticos relativos à incidência de vírus que provoquem encefalites e meningites. Existem trabalhos realizados nas décadas de 1960 e 1970 sobre a soroprevalência de arbovírus e de poliovirus. Utilizando a técnica de reação em cadeia da polimerase (PCR) no líquido cefalorraquidiano (LCR) é possível realizar, em pouco tempo, um diagnóstico de certeza sobre diversos agentes virais responsáveis por estas neuroviroses. Material e métodos: buscou-se determinar a incidência de vírus das famílias herpes, enterovírus e grupo arbovírus pelas técnicas de PCR no LCR de pacientes VIH negativos. Resultados: este trabalho apresenta 59 pacientes VIH negativos que tiveram encefalite ou meningite com provável etiologia viral. Estes agentes são responsáveis pelo maior número de casos de meningite e encefalite no nosso continente. Conclusões: em mais da metade dos casos apresentados foi possível realizar o diagnóstico virológico final; registrou-se uma predominância dos vírus da família herpes tanto em crianças como em adultos sendo que a incidência de enterovírus não era desprezível. Não se detectou a presença de arbovirus.

Summary Introduction: in our country there are no systematic studies on the incidence of virus as a cause of encephalitis and meningitis. However, some studies on the seroprevalence of arbovirus and poliovirus were carried out in the sixties and seventies. Today, the polymerase chain reaction (PCR) applied to cerebrospinal fluid technique allows us to obtain accurate diagnosis within a short time, on the different viral agents that cause these neuroviroses. Method: we explored the incidence of virus from the herpesvirus, enterovirus and arbovirus group through PCR techniques applied to HIV negative patients. Results: this study included 59 HIV negative patients who suffered from encephalitis and meningitis of viral etiology. These agents are responsible for most of the cases of meningitis and encephalitis in our continent. Conclusions: the final viral diagnosis may be obtained in over half of the cases presented. The herpesvirus is the most frequent both in children and in adults, being it significant the incidence of enterovirus. No arbovirus were identified in this study.

3613 downloads

Serologic evidence of human metapneumovirus circulation in Uruguay
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Mirazo, Santiago

; Ruchansky, Dora

; Blanc, Andrea

; Arbiza, Juan

Memórias do Instituto Oswaldo Cruz

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nov 2005, Volume 100 N. 7 Pages 715 - 718

Abstract: En | Text: En | PDF: En

First identified in 2001, the human metapneumovirus (hMPV), is a respiratory tract pathogen that affects young children, elderly, and immunocompromised patients. The present work represents the first serologic study carried out in Uruguay. It was performed with the purpose of obtaining serological evidence of hMPV circulation in Uruguay and to contribute to the few serologic reports described until now. Sixty nine serum samples collected between 1998 and 2001 by vein puncture from patients without respiratory symptoms or underlying pathology aged 6 days to 60 years were examined using an indirect immunofluorescence assay (IFA). The global seropositivity rate of the samples was 80% (55/69). Rates of 60% (15/25) and 91% (40/44) were observed for the pediatric and adult cohorts, respectively. Results obtained from a longitudinal analysis of 6 children aged 6 days to 18 months are discussed. These results are a clear evidence of hMPV circulation in Uruguay, at least since 1998, and reinforce the previous data on worldwide circulation of this virus.

1719 downloads Cited 3 times in SciELO

Molecular epidemiology of human respiratory syncytial virus in Uruguay: 1985-2001 - A review
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Arbiza, Juan

; Delfraro, Adriana

; Frabasile, Sandra

Memórias do Instituto Oswaldo Cruz

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may 2005, Volume 100 N. 3 Pages 221 - 230

Abstract: En | Text: En | PDF: En

The variability of the G glycoprotein from human respiratory syncytial viruses (HRSV) (groups A and B) isolated during 17 consecutive epidemics in Montevideo, Uruguay have been analyzed. Several annual epidemics were studied, where strains from groups A and B circulated together throughout the epidemics with predominance of one of them. Usually, group A predominates, but in some epidemics group B is more frequently detected. To analyse the antigenic diversity of the strains, extracts of cells infected with different viruses of group A were tested with a panel of anti-G monoclonal antibodies (MAbs). The genetic variability of both groups was analyzed by sequencing the C-terminal third of the G protein gene. The sequences obtained together with previously published sequences were used to perform phylogenetic analyses. The data from Uruguayan isolates, together with those from the rest of the world provide information regarding worldwide strain circulation. Phylogenetic analyses of HRSV from groups A and B show a model of evolution analogous to the one proposed for influenza B viruses providing information that would be beneficial for future immunization programs and to design safe vaccines.

2229 downloads Cited 2 times in SciELO

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