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2.
Chinese Journal of Biotechnology ; (12): 1269-1276, 2020.
Article in Chinese | WPRIM | ID: wpr-826850

ABSTRACT

Human adenoviruses are widespread causative agent that induces respiratory diseases, epidemic keratoconjunctivitis and other related diseases. Adenoviruses are commonly used in experimental and clinical areas. It is one of the most commonly used virus vectors in gene therapy, and it has attracted a lot of attention and has a high research potential in tumor gene therapy and virus oncolytic. Here, we summarize the biological characteristics, epidemiology and current application of adenovirus, in order to provide reference for engineering application of adenovirus.


Subject(s)
Adenovirus Infections, Human , Epidemiology , Virology , Adenoviruses, Human , Genetics , Genetic Engineering , Methods , Genetic Vectors , Humans , Oncolytic Virotherapy , Oncolytic Viruses , Genetics , Virus Replication
3.
Rio de Janeiro; s.n; 2020. 157 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-1177895

ABSTRACT

Neste estudo avaliamos o impacto das doenças diarreicas agudas (DDA) associadas aos rotavírus A (RVA), norovírus, adenovírus (HAdV), sapovírus (SaV), bocavírus (HBoV) e astrovírus (HAstV) em crianças ≤ 5 anos atendidas na emergência do Hospital da Criança de Santo Antônio (HCSA), Boa Vista, Roraima (RR), no período de outubro de 2016 a outubro de 2017. Foram coletadas, paralelamente, amostras de fezes e de saliva de 734 crianças sendo 485 com DDA e 249 com infecção respiratória aguda (IRA, grupo controle). O estudo teve a aprovação do CEP:1.333.480, 23/11/2015, UFRR. Os RVA, norovírus, HAdV, SaV e HBoV foram pesquisados nas 734 amostras de fezes e HAstV em 170 amostras de crianças com DDA. Adicionalmente, a presença dos HBoV foi também investigada em 38 amostras de saliva sendo 25 de crianças com DDA e 13 IRA. O perfil de susceptibilidade AB0, Lewis e secretor dos antígenos do grupo histosanguíneo (HBGA) foi determinado para todas as 734 crianças em salivas pela fenotipagem e em amostras de salivas pela genotipagem, sendo para o gene FUT2 em 166 amostras e para FUT3 em 42 amostras. Os aspectos clínicos, epidemiológicos e a cobertura da vacina Rotarix® (RV1) foram também avaliados. Os RVA, norovírus e SaV foram investigados pela metodologia de transcrição reversa seguida de amplificação genômica quantitativa (RT-qPCR); os HBoV e HAdV pela amplificação genômica quantitativa (qPCR) e os HAstV por amplificação genômica qualitativa (RT-PCR). A genotipagem dos HBoV e a detecção/genotipagem dos HAstV foi realizada pela PCR seguida de sequenciamento nucleotídico (método Sanger). O Ensaio imunoenzimático (EIA) e a amplificação genômica específica (PCR-touchdown), seguida de sequenciamento nucleotídico (Sanger) foram utilizadas respectivamente para a fenotipagem e genotipagem dos HBGA.


Nas crianças com DDA (n=485), observou-se as seguintes frequências virais: RVA (22,7%), norovírus (38%), HAdV (33,6%), SaV (7,3%) e HBoV (14,2%). Nas crianças com IRA (n=249), observou as seguintes frequências: RVA (19,3%), norovírus (21,3%), HAdV (39,5%), SaV (5,6%) e HBoV (14,1%). O perfil de detecção do HBoV nas 76 amostras de saliva e fezes pareadas foi diferente e correlacionado com os genótipos 1 a 3 detectados. Quanto aos HAstV, nas 170 amostras investigadas, observou-se as seguintes frequências e genótipos: HAstV clássicos: HAstV3 (0,60%) e HAsV5 (1,8%); HAstV não clássicos: HAstVMLB1-2 (3,5%), sendo esta a primeira descrição do HAstVMLB2 no Brasil. A cobertura vacinal para RV1 calculada foi de 61% e crianças vacinadas na faixa etária entre 6 e 24 meses apresentaram frequências mais elevadas de infecção pelo RVA. As 734 crianças apresentaram majoritariamente (54.5%) o perfil Lea+b+ (fraco secretor) e polimorfirmos de nucleotídico único (SNPs) foram detectados nos genes FUT2 e FUT3. Foi detectada susceptibilidade (HBGA) para a infecção pelos HAdV em crianças com IRA, perfil fraco secretor e grupo sanguíneo A ou O, de acordo com valores de Odds Ratio (OR) calculado. As frequência dos vírus detectados principalmente para os norovírus e HAdV em crianças com DDA, demostram a importância da etiologia viral nas DDA em crianças ≤ 5 anos de idade no período do estudo e podem estar relacionadas ao fator de susceptibilidade ao HBGA (incluindo heterogeneidade genética) e a baixa cobertura de RV1. (AU)


Subject(s)
Humans , Child, Preschool , Rotavirus Infections , Adenoviruses, Human , Child, Preschool , Caliciviridae Infections , Sapovirus , Dysentery , Avastrovirus
4.
Neumol. pediátr. (En línea) ; 14(1): 12-18, abr. 2019. ilus, tab
Article in Spanish | LILACS | ID: biblio-995613

ABSTRACT

Acute respiratory infections represent a world pediatric health burden. RSV, influenza and adenoviruses are among the most frequent causative agents. Adenoviruses usually produce upper respiratory infections, but they can be responsible for acute lower respiratory infection in children with severe clinical outcome. It is necessary a special clinical and epidemiological organization to avoid nosocomial adenovirus local outbreaks. Rapid diagnose, done by immunofluorescence assay and PCR, individual case isolation and supportive therapy are necessary for an appropriate management. The increasing immune compromised population represents a challenge for the adenovirus diagnosis with quantitative PCR and for nosocomial infection control and potential antiviral treatment.


Las infecciones respiratorias agudas son un problema prioritario mundial de morbimortalidad infantil y son causadas predominantemente por virus, entre los que destacan el virus respiratorio sincicial (VRS), virus influenza (FLU) y adenovirus (ADV). Los ADV normalmente causan infecciones respiratorias altas, pero pueden provocar infecciones bajas muy graves, que dejan secuelas y tienen alta letalidad. Requieren un manejo clínico y epidemiológico especial para evitar los graves brotes nosocomiales observados en Latinoamérica. Esto incluye un diagnóstico rápido hecho con técnicas de inmunodiagnóstico y reacción en cadena polimerasa (PCR), aislamiento individual del paciente y terapia de soporte. En pacientes inmunocomprometidos, la infección por ADV representa un gran desafío para el diagnóstico, con uso de PCR cuantitativo (qPCR) y eventual tratamiento antiviral. El objetivo de esta revisión es el de actualizar las propiedades, patogenia, epidemiología y diagnóstico del ADV, con énfasis en los cuadros respiratorios de mayor morbimortalidad que se producen en algunos niños.


Subject(s)
Humans , Infant , Child , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/etiology , Adenovirus Infections, Human/diagnosis , Adenovirus Infections, Human/etiology , Respiratory Tract Infections/therapy , Respiratory Tract Infections/epidemiology , Respiratory Tract Infections/virology , Adenovirus Infections, Human/therapy , Adenovirus Infections, Human/epidemiology , Adenoviruses, Human/pathogenicity
5.
Article in Chinese | WPRIM | ID: wpr-775558

ABSTRACT

Human adenovirus (HAdV) is one of the common pathogens causing human respiratory infections and HAdV infection can result in a variety of diseases. In recent years, outbreaks of HAdV infection have been detected from time to time in China and clustered severe cases have been reported in some regions. This technical guideline has been timely developed to provide technical support on the control and prevention of HAdV respiratory infections. It provides an overview of etiology, epidemiology, clinical manifestations, and treatment principles of HAdV infection, determines the definitions of laboratory-confirmed cases, clinically diagnosed cases, severe and critically severe HAdV pneumonia cases. Then the workflow of case detecting and reporting, and the outbreak epidemic disposal has been formulated. Finally, the control and prevention measures in places at high risk for HAdV transmission and individual preventive measures also has been introduced.


Subject(s)
Adenovirus Infections, Human , Virology , Adenoviruses, Human , China , Humans , Practice Guidelines as Topic , Respiratory Tract Infections , Virology
6.
Rev. Soc. Bras. Med. Trop ; 51(1): 30-38, Jan.-Feb. 2018. tab, graf
Article in English | LILACS, ColecionaSUS, CONASS, SES-RS | ID: biblio-897050

ABSTRACT

INTRODUCTION Infections caused by respiratory viruses are important problems worldwide, especially in children. Human metapneumovirus (hMPV) is a respiratory pathogen and causes severe infections with nonspecific symptoms. This study reports the hMPV occurrence and dissemination in southern Brazil and compares the frequency of occurrence of this virus and the human respiratory syncytial virus (hRSV) in the epidemiological weeks in a three-year period (2009-2011). METHODS: In total, 545 nasopharyngeal (NP) specimens from individuals with Severe Acute Respiratory Syndrome (SARS) who were negative for other seven respiratory viruses were analyzed for the presence of hMPV. Human metapneumovirus was detected by direct immunofluorescence and real-time reverse transcription polymerase chain reaction. RESULTS: hMPV was detected in 109 patients from the main geographic regions of the southernmost state of Brazil, presenting similar overall prevalence in males (46.8%) and females (53.2%). Among children who were less than six years old, hMPV was detected in 99 samples of all age groups, with a higher frequency in infants who were less than one year old (45.7%) compared to all other age groups until six years. hMPV and hRSV infection occurred in almost the same epidemiological weeks (EWs) of each year, with peaks of incidence between EW 31/37 and EW 26/38 for the years 2009 and 2011, respectively. hMPV was further detected in several cases of SARS and it was the only virus detected in three deaths. CONCLUSIONS These findings indicate that hMPV is in circulation in southern Brazil and highlight the importance of diagnosing hMPV for influenza-like illness in the population. (AU)


Subject(s)
Humans , Male , Female , Pregnancy , Infant, Newborn , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Respiratory Tract Infections/transmission , Respiratory Tract Infections/virology , Respiratory Syncytial Virus Infections/virology , Metapneumovirus/pathogenicity , Epidemiological Monitoring , Adenoviruses, Human , Pneumovirinae/classification , Paramyxoviridae Infections/virology , Coronavirus , Enterovirus , Severe Acute Respiratory Syndrome , Influenza, Human , Human bocavirus
8.
Article in English | WPRIM | ID: wpr-715376

ABSTRACT

Epidemic keratoconjunctivitis (EKC) and acute hemorrhagic conjunctivitis (AHC) are common diseases caused by human adenoviruses (HAdV) and enteroviruses, respectively, in South Korea. However, there are limited studies on the molecular epidemiology of viral conjunctivitis in South Korea. The main objective of this study was to characterize the genotypes of adenoviruses and enteroviruses causing viral conjunctivitis in the southwest region of South Korea. We collected conjunctival swabs from 492 patients with suspected cases of viral conjunctivitis from 6 ophthalmic hospitals in Gwangju Metropolitan City, in South Korea, between 2012 and 2016. Of the 492 samples tested, HAdVs and enteroviruses were detected in 249 samples (50.6%) and 19 samples (3.9%), respectively. The genotype analysis detected HAdV-8 in 183 samples (73.5%), HAdV-37 in 14 samples (5.6%), and HAdV-3, and HAdV-4 in 9 samples (3.6%) each. We detected coxsackievirus A24 (CVA24) and coxsackievirus B1 (CVB1) in 8 samples (42.0%) and 4 samples (21.0%), respectively. We also reported for the first time HAdV-56-infected cases of EKC in South Korea. Furthermore, we found three cases of coinfection with HAdV and enterovirus genotypes in our samples. HAdV-8 and CVA24, the main causes of EKC and AHC, respectively, worldwide, were also found to be the predominant genotypes in our study.


Subject(s)
Adenoviridae , Adenoviruses, Human , Coinfection , Conjunctivitis, Acute Hemorrhagic , Conjunctivitis, Viral , Enterovirus , Genotype , Humans , Keratoconjunctivitis , Korea , Molecular Epidemiology
9.
Article in Korean | WPRIM | ID: wpr-718761

ABSTRACT

Respiratory infections, which are caused by airborne pathogens, are the most common disease of all ages worldwide. This study was conducted to characterize the airborne respiratory pathogens in the public facilities in Busan, South Korea. A total of 260 public facilities were investigated in 2017, 52 seasonal indoor air from 2 hospitals and 208 indoor air samples from 208 randomly selected daycare centers. Among respiratory pathogen, 8 viral pathogens including human adenovirus (HAdV), human bocavirus (HBoV), human rhinovirus (HRV), human parainfluenza virus (HPIV), human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), human coronavirus (HCoV) and influenza virus (IFV), and 3 bacterial pathogens including Mycoplasma pneumoniae, Bordetella pertussis, and Chlamydophila pneumoniae, were investigated by multiplex real-time reverse transcription polymerase chain reaction. Pathogens were detected in 9 cases (3.4%). Among 9 positive samples, 6 (2.3%) cases were positive for HBoV and 3 (1.2%) cases were positive for IFV. All the positive cases were detected in daycare centers. Additionally, the concentration of HBoV was determined. In HBoV-positive samples, the cycle threshold (Ct) values of HBoV were 29.73~36.84, which are corresponding to the viral concentration of 4.91 × 10⁰ ~ 9.57 × 10² copies/ml. Serotype distribution of isolated HBoV was analyzed by sequencing of VP1/VP2 gene. All of the HBoV isolates were identified as HBoV type 1 with a high similarity among the isolates (>97%). No bacterial pathogen was identified in indoor air samples. Although virus concentration was not high in public facilities (daycare center), the presence of respiratory viral pathogens has been identified. Effective ventilation and air purification strategies are needed to reduce the indoor concentration of respiratory pathogens. A long-term and ongoing surveillance plan for respiratory pathogen management should be established.


Subject(s)
Adenoviruses, Human , Bordetella pertussis , Chlamydial Pneumonia , Chlamydophila pneumoniae , Coronavirus , Human bocavirus , Humans , Korea , Metapneumovirus , Mycoplasma pneumoniae , Orthomyxoviridae , Paramyxoviridae Infections , Pneumonia, Mycoplasma , Polymerase Chain Reaction , Public Facilities , Respiratory Syncytial Virus, Human , Respiratory Tract Infections , Reverse Transcription , Rhinovirus , Seasons , Serogroup , Ventilation
10.
Braz. j. infect. dis ; 21(5): 500-506, Sept.-Oct. 2017. tab, graf
Article in English | LILACS | ID: biblio-888912

ABSTRACT

Abstract Human adenoviruses comprise an important group of etiologic agents that are responsible for various diseases in adults and children, such as respiratory, ocular, gastroenteric, and urinary infections. In immunocompromised and organ-transplanted individuals, these agents can cause generalized infections. Rapid diagnostic methods for detecting these infectious agents are not widely available.The aim of this work was to produce monoclonal and polyclonal anti-adenovirus antibodies to be used in a rapid diagnostic test for respiratory infections.Adenovirus hexons were satisfactorily purified by ultracentrifugation and chromatography. After virus purification, anti-hexon monoclonal antibodies were produced and characterized, following classical methods. Antibodies were specific for adenoviruses 2, 3, 5, and 41. The proposed immunochromatographic test was standardized using colloidal gold.The standardization of the rapid test was sufficient to detect adenovirus antigens (in nasopharyngeal lavage samples) with sensitivity of 100% and specificity of 85% when compared to direct immunofluorescence.The immunochromatographic assay prototype was sufficiently sensitive to detect B (3), C (2 and 5), and F (41) adenovirus samples. Although based on preliminary data, the test demonstrated the same performance as direct immunofluorescence, but with the advantage of being a point-of-care test. Further studies are still needed to confirm its effectiveness in clinical practice.


Subject(s)
Humans , Adenoviruses, Human/classification , Chromatography, Affinity/methods , Adenoviridae Infections/diagnosis , Adenoviridae Infections/virology , Antibodies, Viral/blood
11.
Rev. Soc. Bras. Med. Trop ; 50(5): 621-628, Sept.-Oct. 2017. tab, graf
Article in English | LILACS | ID: biblio-897017

ABSTRACT

Abstract INTRODUCTION: Acute gastroenteritis (AGE) is one of the most common causes of morbidity and mortality, especially among children from developing countries. Human adenovirus (HAdV) and sapovirus (SaV) are among the agents that cause AGE. The present study aimed to detect and genotype HAdV and SaV in 172 fecal samples from children with AGE, collected during a surveillance study carried out in a low-income community in Belém, Pará, between 1990 and 1992. METHODS: HAdV was detected by nested PCR, using primers Hex1deg/Hex2deg and NeHex3deg/NeHex4deg. SaV was assayed by reverse transcription PCR (RT-PCR), nested PCR, and quantitative PCR. The nucleotide sequence was determined by direct cycle sequencing. RESULTS: Overall, 43% (74/172) of samples were positive for HAdV, of which 70.3% (52/74) were sequenced and classified as belonging to five different species, mostly A and F. For SaV, positivity was 5.2% (9/172) and genotypes GI.1, GI.7, GII.1, and GV.2 were detected. CONCLUSIONS: The present results reinforce the need for further studies to obtain epidemiological data about the circulation of these viruses in Brazil, especially in the Amazon Region, where data from the early 1990's are scarce. Furthermore, the study describes for the first time the detection of SaV genotypes GI.7 and GV.2 in Brazil, showing that these types circulated in the region more than 25 years ago.


Subject(s)
Humans , Male , Female , Infant, Newborn , Infant , Child, Preschool , Brazil/epidemiology , Adenoviruses, Human/isolation & purification , Caliciviridae Infections/virology , Sapovirus/isolation & purification , Gastroenteritis/virology , Genotype , Phylogeny , Time Factors , Base Sequence , Adenovirus Infections, Human/epidemiology , Adenovirus Infections, Human/virology , Adenoviruses, Human/genetics , Polymerase Chain Reaction , Prevalence , Prospective Studies , Age Distribution , Caliciviridae Infections/epidemiology , Sapovirus/genetics , Genotyping Techniques/methods , Gastroenteritis/enzymology , Genes, Viral
13.
Article in English | WPRIM | ID: wpr-109776

ABSTRACT

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.


Subject(s)
Adenoviridae , Adenoviruses, Human , Biology , Codon, Initiator , Genetic Therapy , Genome , Genome, Viral , Leucine Zippers , Species Specificity , Transfection , Virion
14.
Braz. j. microbiol ; 47(1): 243-250, Jan.-Mar. 2016. tab, graf
Article in English | LILACS | ID: lil-775111

ABSTRACT

Abstract Human adenovirus species F (HAdV-F) type 40 and 41 are commonly associated with acute diarrheal disease (ADD) across the world. Despite being the largest state in southeastern Brazil and having the second largest number of inhabitants, there is no information in the State of Minas Gerais regarding the role of HAdV-F in the etiology of ADD. This study was performed to determine the prevalence, to verify the epidemiological aspects of infection, and to characterize the strains of human adenoviruses (HAdV) detected. A total of 377 diarrheal fecal samples were obtained between January 2007 and August 2011 from inpatient and outpatient children of age ranging from 0 to 12 years. All samples were previously tested for rotavirus, norovirus, and astrovirus, and 314 of 377 were negative. The viral DNA was extracted, amplified using the polymerase chain reaction and the HAdV-positive samples were sequenced and phylogenetically analyzed. Statistical analyses were performed using the Chi-square test (p < 0.05), considering two conditions: the total of samples tested (377) and the total of negative samples for the remaining viruses tested (314). The overall prevalence of HAdV was 12.47% (47/377); and in 76.60% (36/47) of the positive samples, this virus was the only infectious agent detected. The phylogenetic analysis of partial sequences of 32 positive samples revealed that they all clustered with the HAdV-F type 41. The statistical analysis showed that there was no correlation between the onset of the HAdV infection and the origin of the samples (inpatients or outpatients) in the two conditions tested: the total of samples tested (p = 0.598) and the total of negative samples for the remaining viruses tested (p = 0.614). There was a significant association in the occurrence of infection in children aged 0–12 months for the condition 1 (p = 0.030) as well as condition 2 (p = 0.019). The occurrence of infections due to HAdV did not coincide with a pattern of seasonal distribution. These data indicate the significant involvement of HAdV-F type 41 in the etiology of ADD in Minas Gerais, which demonstrates the importance of other viral agents in the development of the disease after the introduction of rotavirus vaccine immunization.


Subject(s)
Child , Child, Preschool , Humans , Infant , Infant, Newborn , Adenovirus Vaccines/administration & dosage , Adenoviruses, Human/isolation & purification , Diarrhea/epidemiology , Diarrhea/prevention & control , Rotavirus Infections/epidemiology , Rotavirus Infections/prevention & control , Adenovirus Vaccines/immunology , Adenoviruses, Human/classification , Adenoviruses, Human/genetics , Brazil/epidemiology , Cluster Analysis , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/isolation & purification , Feces/virology , Genotype , Phylogeny , Prevalence , Sequence Analysis, DNA
15.
Mundo saúde (Impr.) ; 40(4): 474-480, nov. 2016. ilus
Article in Portuguese | LILACS | ID: biblio-996865

ABSTRACT

Com o desenvolvimento humano, os recursos hídricos sofrem modificações derivadas da expansão demográfica e principalmente da falta de saneamento básico, que interferem diretamente na qualidade desses recursos com a introdução de substâncias químicas tóxicas e bioagentes patogênicos. Os vírus entéricos de veiculação hídrica, como adenovírus e rotavírus, merecem destaque considerando seus impactos na saúde pública. O objetivo deste estudo foi avaliar a presença de adenovírus e rotavírus em águas superficiais do Córrego Ribeirão Preto, SP. A coleta foi realizada em 11 pontos distintos, da nascente até a confluência com o Rio Pardo. O Teste de ELISA foi o método utilizado para a detecção dos vírus (RIDASCREEN® Adenovirus, RIDASCREEN® Rotavirus - R-Biopharm). De acordo com os resultados obtidos, 73% das amostras foram positivas para adenovírus, enquanto que 36% foram positivas para rotavírus. Considerando a virulência desses vírus entéricos e a vulnerabilidade de indivíduos imunossuprimidos, idosos e crianças, é preocupante a contaminação por esses agentes patogênicos de veiculação hídrica amplamente disseminados em águas superficiais.


Water resources have been suffering with the changes in demographic expansion and the lack of basic sanitation. These factors directly interfere with the quality of these resources due to the discharge of toxic chemicals and pathogenic bio-agents. The waterborne enteric viruses, such as rotavirus and adenovirus, deserve attention considering its impact on public health. The aim of this study was to evaluate the presence of adenovirus and rotavirus in surface water of Ribeirão Preto stream. Samplings were conducted in 11 different points from the spring to the confluence with the Rio Pardo. The ELISA test was carried out for the detection of viruses (RIDASCREEN® adenovirus, rotavirus RIDASCREEN® - R-Biopharm). According to the results, 73% of the samples were positive for adenovirus, while 36% were positive for rotavirus. Given the virulence of these enteric viruses and the vulnerability of immunosuppressed individuals, i.e. the elderly and children, contamination with these waterborne pathogens widely disseminated in surface water is concerning


Subject(s)
Humans , Surface Waters , Adenoviruses, Human , Sanitation , Environmental Health , Rotavirus , Quality of Life , Public Health
16.
Article in Chinese | WPRIM | ID: wpr-263999

ABSTRACT

<p><b>OBJECTIVE</b>To construct a recombinant human adenovirus type 5 (Ad5) expressing luciferase and GFP reporter gene and detect neutralizing antibodies against adenovirus type 5 in common marmosets (Callithrix jacchus) to provide basic laboratory data for evaluating adenovirus vaccines.</p><p><b>METHODS</b>Luciferase and GFP reporter genes from plasmid pHAGE-CMV-GFP were inserted into pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-GFP. The shutter plasmid was co-transduced with pBHGlox(delta)E1,3Cre in 293A cell line to package the recombinant adenovirus rAd5/Luc/GFP. Three rounds of plaque formation experiment were performed to select the monoclonal adenovirus followed by purification with cesium chloride density gradient centrifugation and virus titration with TCID50 method. Chemiluminescence assay and flow cytometry were employed to detect the neutralizing antibody levels in 14 common marmosets.</p><p><b>RESULTS</b>The shuttle plasmid pDC315-Luc-GFP was successfully constructed and the recombinant adenovirus rAd5/Luc/GFP was packaged with a the titer reaching 6.9×10(11.5) PFU/mL. In the 14 marmosets, chemiluminescence assay identified 4 (28.6%) marmosets that were positive for Ad5-neutralizing antibodies, including 2 with a antibody titer of 1/16 and another 2 with a titer of 1/32; flow cytomery detected Ad5-neutralizing antibodies in 3 marmosets at the titer of 1/16.</p><p><b>CONCLUSION</b>Chemiluminescence assay is a simple, sensitive, and accurate modality for detecting Ad5-neutralizing antibodies. Common marmosets have a very low positivity rate for Ad5-neutralizing antibodies and are therefore promising models for studying adenovirus-based vaccines and therapies.</p>


Subject(s)
Adenoviruses, Human , Allergy and Immunology , Animals , Antibodies, Neutralizing , Blood , Antibodies, Viral , Blood , Callithrix , Cell Line , Humans , Immunity, Humoral , Luciferases , Plasmids
17.
Chinese Journal of Virology ; (6): 32-38, 2016.
Article in Chinese | WPRIM | ID: wpr-296220

ABSTRACT

We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.


Subject(s)
Adenoviruses, Human , Chemistry , Genetics , Metabolism , Arabidopsis Proteins , Chemistry , Genetics , Metabolism , Flavoproteins , Chemistry , Genetics , Metabolism , Humans , Phototropins , Chemistry , Genetics , Metabolism , Singlet Oxygen , Chemistry , Staining and Labeling , Transfection
18.
Braz. j. biol ; 75(4,supl.2): 37-42, Dec. 2015. tab, graf
Article in English | LILACS | ID: lil-769600

ABSTRACT

The present study analyzed the efficiency of the photo-electro-oxidation process as a method for degradation and inactivation of adenovirus in water. The experimental design employed a solution prepared from sterile water containing 5.107 genomic copies/L (gc/L) of a standard strain of human adenovirus type 5 (HAdV-5) divided into two equal parts, one to serve as control and one treated by photo-electro-oxidation (PEO) for 3 hours and with a 5A current. Samples collected throughout the exposure process were analyzed by real-time polymerase chain reaction (qPCR) for viral genome identification and quantitation. Prior to gene extraction, a parallel DNAse treatment step was carried out to assess the integrity of viral particles. Integrated cell culture (ICC) analyses assessed the viability of infection in a cell culture. The tested process proved effective for viral degradation, with a 7 log10 reduction in viral load after 60 minutes of treatment. The DNAse-treated samples exhibited complete reduction of viral load after a 75 minute exposure to the process, and ICC analyses showed completely non-viable viral particles at 30 minutes of treatment.


Resumo O presente estudo analisou a eficiência do processo de fotoeletrooxidação como metodologia para a degradação e inativação de adenovírus em água. A concepção experimental emprega uma solução preparada a partir de água estéril contendo 5,107 cópias genômicas/L (gc/L) de uma amostra padrão de adenovírus humano tipo 5 (HAdV-5), dividida em duas partes iguais, uma para servir como controle e outra tratada por fotoeletrooxidação (PEO) durante 3 horas e com uma corrente de 5A. As amostras recolhidas durante o processo de exposição foram analisadas por PCR quantitativo em tempo real (qPCR) para identificação e quantificação do genoma viral. Antes da extração de ácidos nucleicos, um passo de tratamento com DNAse paralelo foi realizado para avaliar a integridade das partículas virais. Um ensaio de qPCR integrado à cultura de células (ICC-qPCR) permitiu analisar a viabilidade de infecção em uma cultura de células. O processo mostrou-se eficaz testada para a degradação viral, com uma redução de 7 log10 da carga viral após 60 minutos de tratamento. As amostras tratadas com DNAse exibiram redução completa da carga viral após uma exposição de 75 minutos ao processo, e a análise de ICC-qPCR mostrou partículas virais completamente não-viáveis ​​em 30 minutos de tratamento.


Subject(s)
Adenoviruses, Human/isolation & purification , Virus Inactivation , Waste Disposal, Fluid/methods , Water Purification/methods , Electrochemical Techniques , Oxidation-Reduction , Photolysis , Real-Time Polymerase Chain Reaction
19.
Braz. j. microbiol ; 46(3): 749-752, July-Sept. 2015.
Article in English | LILACS | ID: lil-755826

ABSTRACT

Human adenoviruses (HAdV), members of the Adenoviridae family, are excreted through the fecal route and may be present in the feces of humans consuming contaminated food or water. The presence of HAdV from different serotypes in the feces of healthy individuals was already reported using conventional polymerase chain reaction; however, real-time PCR (qPCR) may reveal not only the rates of detection as well as demonstrate the viral loads excreted by healthy persons. Aiming to identify and characterize the presence of adenoviruses in stool samples, 147 fecal samples from patients with no records of diarrhea were analyzed (74 from winter season and 73 from summer) by Real-Time PCR (qPCR) assay and conventional PCR. HAdV genome was present in 43.8% (32/73) of stools samples collected during summer season and 21.6% (16/74) during winter. The rate of detection of genomic copies (gc) ranged from 4.04×102 to 6.72×105gc/g of feces among the 147 samples analyzed, of which the ranged of genomic copies of DNA HAdV was major in summer. All samples were negative when tested for rotaviruses (RV) and noroviruses (NoV) by PCR conventional and qPCR respectively. HAdV is excreted constantly by infected individuals in the absence of clinical signs and the occurrence may vary seasonally.

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Subject(s)
Adult , Female , Humans , Male , Middle Aged , Young Adult , Adenovirus Infections, Human/epidemiology , Adenoviruses, Human/genetics , Adenoviruses, Human/isolation & purification , Feces/virology , Adenovirus Infections, Human/virology , Adenoviruses, Human/classification , Brazil , Norovirus/genetics , Norovirus/isolation & purification , Real-Time Polymerase Chain Reaction , Rotavirus/genetics , Rotavirus/isolation & purification , Seasons
20.
Rev. Inst. Med. Trop. Säo Paulo ; 57(4): 299-303, July-Aug. 2015. tab
Article in English | LILACS | ID: lil-761160

ABSTRACT

SUMMARYHuman Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.


RESUMOOs adenovírus humanos (HAdV) são notavelmente resistentes ao ambiente. Estes agentes podem servir como indicadores efetivos de contaminação fecal, tanto quanto podem atuar como agentes causadores de diferentes doenças em seres humanos. A reação em cadeia da polimerase (PCR) e mais recentemente a PCR quantitativa (qPCR) são amplamente usadas para detecção de agentes virais em matrizes ambientais. No presente estudo, PCR e SYBR(r)Green qPCR foram comparadas para a detecção de HAdV em amostras de água (55) e sedimento (20) provenientes de nascentes, poços, açudes e arroios coletadas em propriedades leiteiras. A metodologia quantitativa detectou HAdV em 87,3% das amostras de água e 80% dos sedimentos, enquanto por PCR convencional a detecção foi de 47,3% e 35%, respectivamente.


Subject(s)
Adenoviruses, Human/isolation & purification , Geologic Sediments/virology , Polymerase Chain Reaction/methods , Water Microbiology , Environmental Monitoring
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