Development of a prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus infection

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.

Development of a prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus infection.

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.

Development of a prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus infection

Human adenoviruses comprise an important group of etiologie 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.& para;& para;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.& para;& para;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.& para;& para;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.& para;& para;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.

Generation and biological properties of a recombinant dodecahedron containing the short fiber protein of the human adenovirus 41.

OBJECTIVE: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. METHODS: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five™ cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. RESULTS: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. CONCLUSION: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification.

Gravidade das coinfecções virais em lactentes hospitalizados com infecção por vírus sincicial respiratório / Severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection

OBJETIVO: Comparar a gravidade de infecções causadas por um único vírus (VSR) com a gravidade de coinfecções. MÉTODOS: Este estudo avaliou uma coorte histórica de lactentes com infecção aguda por VSR. Secreção de nasofaringe foi coletada de todos os pacientes rotineiramente para pesquisa viral usando técnicas de biologia molecular. Os seguintes desfechos foram analisados: tempo total de internação, duração da oxigenioterapia, admissão em unidade de terapia intensiva e uso de ventilação mecânica. Os resultados foram ajustados para os fatores confundidores (prematuridade, idade e aleitamento materno). RESULTADOS: Foram incluídos no estudo 176 lactentes com idade média de 4,5 meses e diagnósticos de bronquiolite e/ou pneumonia. Cento e vinte e um tinham infecção única por VSR, e 55 tinham coinfecções (24 VSR + adenovírus, 16 VSR + metapneumovírus humano e 15 outras associações menos frequentes). Os quatro desfechos de gravidade avaliados foram semelhantes entre o grupo com infecção única por VSR e os grupos com coinfecções, independente do tipo de vírus associado com o VSR. CONCLUSÃO: As coinfecções virais não parecem alterar o prognóstico de lactentes hospitalizados com infecção aguda por VSR.

OBJECTIVE: To compare the severity of single respiratory syncytial virus (RSV) infections with that of coinfections. METHODS: A historical cohort was studied, including hospitalized infants with acute RSV infection. Nasopharyngeal aspirate samples were collected from all patients to detect eight respiratory viruses using molecular biology techniques. The following outcomes were analyzed: duration of hospitalization and of oxygen therapy, intensive care unit admission and need of mechanical ventilation. Results were adjusted for confounding factors (prematurity, age and breastfeeding). RESULTS: A hundred and seventy six infants with bronchiolitis and/or pneumonia were included in the study. Their median age was 4.5 months. A hundred and twenty one had single RSV infection and 55 had coinfections (24 RSV + adenovirus, 16 RSV + human metapneumovirus and 15 other less frequent viral associations). The four severity outcomes under study were similar in the group with single RSV infection and in the coinfection groups, independently of what virus was associated with RSV. CONCLUSION: Virus coinfections do not seem to affect the prognosis of hospitalized infants with acute RSV infection.

Severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection.

OBJECTIVE: To compare the severity of single respiratory syncytial virus (RSV) infections with that of coinfections. METHODS: A historical cohort was studied, including hospitalized infants with acute RSV infection. Nasopharyngeal aspirate samples were collected from all patients to detect eight respiratory viruses using molecular biology techniques. The following outcomes were analyzed: duration of hospitalization and of oxygen therapy, intensive care unit admission and need of mechanical ventilation. Results were adjusted for confounding factors (prematurity, age and breastfeeding). RESULTS: A hundred and seventy six infants with bronchiolitis and/or pneumonia were included in the study. Their median age was 4.5 months. A hundred and twenty one had single RSV infection and 55 had coinfections (24 RSV + adenovirus, 16 RSV + human metapneumovirus and 15 other less frequent viral associations). The four severity outcomes under study were similar in the group with single RSV infection and in the coinfection groups, independently of what virus was associated with RSV. CONCLUSION: Virus coinfections do not seem to affect the prognosis of hospitalized infants with acute RSV infection.

A naturally occurring human adenovirus type 7 variant with a 1743 bp deletion in the E3 cassette.

Human adenovirus type 7 (HAdV-7) is an important cause of acute respiratory disease (ARD). Different genomic variants of HAdV-7 have been described, designated 7a-7l. In a previous study to investigate risk factors for ARD and wheezing, nasopharyngeal samples were collected from 90 ill children seeking medical attention in Ribeirão Preto, São Paulo, Brazil. HAdVs were identified in 31 samples and were characterized by serum neutralization and genome restriction analysis. Eleven HAdVs were identified as being HAdV-7, five of which were classified as being of genome type 7p (Gomen). Six other HAdV-7 isolates gave new restriction profiles with all enzymes used and were classified as being a new genomic variant, 7m. These isolates were further characterized by sequencing. The hexon and fiber genes of the 7m variant were nearly identical to the prototype, 7p. However, nucleotide sequences from the E3 cassette revealed a 1743 bp deletion affecting the 16.1K, 19K, 20.1K and 20.5K ORFs.

Infection kinetics of human adenovirus serotype 41 in HEK 293 cells.

The purpose of this work was to acquire an overview of the infectious cycle of HAdV-41 in permissive HEK 293 cells and compare it to that observed with the prototype of the genus, Human adenovirus C HAdV-2. HEK 293 cells were infected with each virus separately and were harvested every 12 h for seven days. Infection kinetics were analysed using confocal and electronic microscopy. The results show that, when properly cultivated, HAdV-41 was not fastidious. It had a longer multiplication cycle, which resulted in the release of complete viral particles and viral stocks reached high titres. After 60 h of infection, the export of viral proteins from the infected cell to the extracellular milieu was observed, with a pattern similar to that previously described for HAdV-2 penton-base trafficking after 30 h of infection. HAdV-41 had a non-lytic cycle and the infection spread from the first infected cell to its neighbours. The release process of the viral particles is unknown. The results observed for HAdV-41 infection in HEK 293 cells show how different this virus is from the prototype HAdV-2 and provides information for the development of this vector for use in gene therapy.

Infection kinetics of human adenovirus serotype 41 in HEK 293 cells

The purpose of this work was to acquire an overview of the infectious cycle of HAdV-41 in permissive HEK 293 cells and compare it to that observed with the prototype of the genus, Human adenovirus C HAdV-2. HEK 293 cells were infected with each virus separately and were harvested every 12 h for seven days. Infection kinetics were analysed using confocal and electronic microscopy. The results show that, when properly cultivated, HAdV-41 was not fastidious. It had a longer multiplication cycle, which resulted in the release of complete viral particles and viral stocks reached high titres. After 60 h of infection, the export of viral proteins from the infected cell to the extracellular milieu was observed, with a pattern similar to that previously described for HAdV-2 penton-base trafficking after 30 h of infection. HAdV-41 had a non-lytic cycle and the infection spread from the first infected cell to its neighbours. The release process of the viral particles is unknown. The results observed for HAdV-41 infection in HEK 293 cells show how different this virus is from the prototype HAdV-2 and provides information for the development of this vector for use in gene therapy.

Hantavirus pulmonary syndrome, central plateau, southeastern, and southern Brazil.

Hantavirus pulmonary syndrome (HPS) is an increasing health problem in Brazil because of encroachment of sprawling urban, agricultural, and cattle-raising areas into habitats of subfamily Sigmodontinae rodents, which serve as hantavirus reservoirs. From 1993 through June 2007, a total of 884 cases of HPS were reported in Brazil (case-fatality rate 39%). To better understand this emerging disease, we collected 89 human serum samples and 68 rodent lung samples containing antibodies to hantavirus from a 2,500-km-wide area in Brazil. RNA was isolated from human samples and rodent tissues and subjected to reverse transcription-PCR. Partial sequences of nucleocapsid protein and glycoprotein genes from 22 human and 16 rodent sources indicated only Araraquara virus and Juquitiba virus lineages. The case-fatality rate of HPS was higher in the area with Araraquara virus. This virus, which may be the most virulent hantavirus in Brazil, was associated with areas that have had greater anthropogenic changes.

Duplex-PCR assay for the detection of adenovirus and respiratory syncytial virus in nasopharyngeal samples.

Human adenovirus (HAdV) and human respiratory syncytial virus (HRSV) are important etiologic agents of acute respiratory infections. In this study, a duplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of HAdV and HRSV in clinical samples. Sixty previously screened nasopharyngeal aspirates were used: 20 HAdV-positive, 20 HRSV-positive and 20 double-negative controls. Eight samples were positive for both viruses. The duplex PCR assay proved to be as sensitive and specific as single-target assays and also detected the mixed infections with certainty. The identification of both viruses in a single reaction offers a reduction in both cost and laboratory diagnostic time.

Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira, São Paulo / Estudo longitudinal da ocorrência de adenovírus e vírus da hepatite A em esgoto doméstico na cidade de Limeira, SP

The aim of this study was to verify the presence and annual distribution of adenoviruses and hepatitis A virus in domestic sewage in the city of Limeira, São Paulo. Fifty samples with a volume of 8 liters each were collected weekly from December 2004 to December 2005. The viruses were concentrated by filtration through positively charged ZP60S filter membranes, followed by ultracentrifugation. Human adenoviruses (HAdV) were detected by PCR followed by nested-PCR and screening for species F was done by restriction of the PCR product with TaqI endonuclease. Virus infectivity assays were performed by inoculation of concentrates onto HEp-2 cell monolayers. RT-PCR was used for the detection of hepatitis A virus. HAdV were detected in all samples, and 64% of samples were positive for infectious virus. Species F was present in 82% of the samples. Hepatitis A virus was detected in 48% of the samples. These results demonstrate that HAdV and HAV were present in the domestic sewage of Limeira throughout the period of study, demonstrating the importance of an adequate treatment before the disposal in the environment.

O objetivo do estudo foi verificar a ocorrência e a distribuição anual de adenovírus humanos e vírus da Hepatite A (VHA) no efluente doméstico da cidade de Limeira, São Paulo, ao longo do período de Dezembro de 2004 e Dezembro de 2005, com vistas à futura implementação de sistemas de tratmento de água de esgoto. Cinquenta amostras de efluente bruto com volume de 8L cada foram colhidas semanalmente e os vírus concentrados por filtração em membrana eletropositiva ZP60S, seguida de ultracentrifugação. Adenovírus foram detectados por PCR e nested-PCR. Adenovírus da espécie F foram distinguidos das demais por restrição do produto da PCR com endonuclease TaqI. Ensaios de infectividade viral foram realizados em culturas de células HEp-2. A presença do vírus da hepatite A também foi pesquisada nas mesmas amostras, fazendo-se uso de método de RT-PCR. Adenovírus foram detectados em todas as amostras, sendo a espécie F identificada em 82% destas. Sessenta e quatro por cento dos adenovírus detectados ainda estavam infecciosos. O vírus da Hepatite A foi detectado em 48% das amostras examinadas. Estes resultados evidenciam a presença e a circulação de Adenovírus humano e VHA nas águas de esgoto doméstico de Limeira ao longo do período de estudo, demonstrando a importância de um tratamento adequado desse material antes da disposição no meio ambiente.

Duplex-PCR assay for the detection of adenovirus and respiratory syncytial virus in nasopharyngeal samples

Human adenovirus (HAdV) and human respiratory syncytial virus (HRSV) are important etiologic agents of acute respiratory infections. In this study, a duplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of HAdV and HRSV in clinical samples. Sixty previously screened nasopharyngeal aspirates were used: 20 HAdV-positive, 20 HRSV-positive and 20 double-negative controls. Eight samples were positive for both viruses. The duplex PCR assay proved to be as sensitive and specific as single-target assays and also detected the mixed infections with certainty. The identification of both viruses in a single reaction offers a reduction in both cost and laboratory diagnostic time.

Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira, SÃo Paulo.

The aim of this study was to verify the presence and annual distribution of adenoviruses and hepatitis A virus in domestic sewage in the city of Limeira, São Paulo. Fifty samples with a volume of 8 liters each were collected weekly from December 2004 to December 2005. The viruses were concentrated by filtration through positively charged ZP60S filter membranes, followed by ultracentrifugation. Human adenoviruses (HAdV) were detected by PCR followed by nested-PCR and screening for species F was done by restriction of the PCR product with TaqI endonuclease. Virus infectivity assays were performed by inoculation of concentrates onto HEp-2 cell monolayers. RT-PCR was used for the detection of hepatitis A virus. HAdV were detected in all samples, and 64% of samples were positive for infectious virus. Species F was present in 82% of the samples. Hepatitis A virus was detected in 48% of the samples. These results demonstrate that HAdV and HAV were present in the domestic sewage of Limeira throughout the period of study, demonstrating the importance of an adequate treatment before the disposal in the environment.

Structural and phylogenetic relationship of ORF 31 from the Anticarsia gemmatalis MNPV to poly (ADP-ribose) polymerases (PARP).

ORF 31 is a unique baculovirus gene in the genome of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D). It encodes a putative polypeptide of 369 aa homologous to poly (ADP-ribose) polymerase (PARP) found in the genomes of several organisms. Moreover, we found a phylogenetic association with Group I PARP proteins and a 3D homology model of its conserved PARP C-terminal catalytic domain indicating that had almost an exact spatial superimposition of <1 A with other PARP available structures. The 5' end of ORF 31 mRNA was located at the first nucleotide of a CATT motif at position -27. Using real-time PCR we detected transcripts at 3 h post-infection (p.i.) increasing until 24 h p.i., which coincides with the onset of DNA replication, suggestive of a possible role in DNA metabolism.

Molecular epidemiology of human adenovirus isolated from children hospitalized with acute respiratory infection in São Paulo, Brazil.

In a study of acute respiratory disease, two collections of nasopharyngeal aspirates (NPA) were obtained from children hospitalized at the Pediatric Clinic of the University Hospital, São Paulo, in 1995 and 2000. Adenovirus was detected in 33 (8.2%) of 401 children followed. These viruses were isolated in HEp-2, HEK-293, or NCI-H292 cells and serotyped by neutralization. The genome types were determined after restriction analyses of the genomic DNA extracted from infected cells. Nineteen isolates were characterized as Human adenovirus B, genome types HAdV-3a, HAdV-7h, and HAdV-7h1; 11 as Human adenovirus C, genome types HAdV-1D10, HAdV-2D25, HAdV-5D2, and HAdV-6D3. Our findings show that species C adenoviruses present an endemic infection pattern, with co-circulation of different serotypes and genome types; no new genomic variant was observed. Species B adenoviruses showed epidemic infection patterns, with shifts in the predominant genome type. The isolates from 1995 belong to genome type 7h, or the variant 7h1, with a clear substitution of the type 7b, prevalent in São Paulo for more then 10 years. In 2000, the variant 7h1 predominated and the emergence of the type 3a was observed. Almost 10 years passed between the identification of HAdV-7h in Argentina and its detection in São Paulo. The geographic isolation of these two countries was reduced by the increase in population mobility due to growing commercial relationships.

Risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization.

BACKGROUND: Risk factors for acute wheezing among children in subtropical areas are largely unknown. OBJECTIVE: To investigate the role of viral infections, allergen sensitization, and exposure to indoor allergens as risk factors for acute wheezing in children 0 to 12 years old. METHODS: One hundred thirty-two children 0 to 12 years of age who sought emergency department care for wheezing and 65 children with no history of wheezing were enrolled in this case-control study. Detection of respiratory syncytial virus antigen, rhinovirus and coronavirus RNA, adenovirus, influenza, and parainfluenza antigens was performed in nasal washes. Total IgE and specific IgE to mites, cockroach, cat, and dog were measured with the CAP system. Major allergens from mites, cockroach, cat, and dog were quantified in dust samples by ELISA. Univariate and multivariate analyses were performed by logistic regression. RESULTS: In children under 2 years of age, infection with respiratory viruses and family history of allergy were independently associated with wheezing (odds ratio, 15.5 and 4.2; P = .0001 and P = .008, respectively). Among children 2 to 12 years old, sensitization to inhalant allergens was the major risk factor for wheezing (odds ratio, 2.7; P = .03). High-level allergen exposure, exposure to tobacco smoke, and lack of breast-feeding showed no association with wheezing. CONCLUSIONS: Some risk factors for wheezing previously identified in temperate climates were present in a subtropical area, including respiratory syncytial virus infection in infants and allergy in children older than 2 years. Rhinovirus was not associated with wheezing and did not appear to be a trigger for asthma exacerbations.

Structural studies of human enteric adenovirus type 41.

Enteric adenoviruses of serotypes 40 and 41 possess some specific structural features, one of which is the presence on the virion of two fibers of different lengths and primary sequences. These viruses are notoriously difficult to grow under laboratory conditions. In this paper the successful growth and purification of Ad41 are presented in detail. Structural Ad41 proteins were analyzed by biochemical methods, mass spectrometry, and electron microscopy (EM), in order to identify and localize them on polyacrylamide denaturing gels and to assess the proportion of short and long fibers in the virion. Surprisingly, the three proteins composing virus short and long pentons did not totally enter the denaturing polyacrylamide gels, which is probably due in part to their high pI. The pentons were separately purified and their dimensions were estimated from EM data. The EM images suggest that there are the same amounts of short and long fibers in each virion.

A reaçäo de imunofluorescência indireta no diagnóstico por rotavírus / The indirect immunofluorescence assay in the diagnosis of human rotavirus gastroenteritis

Utilizando a cepa de rotavírus SA-11, cultivada em células MA-104 e purificada em gradiente de CsCl, prepararam-se soros antirotavírus em cobaias para uso no diagnóstico de gastroenterites por rotavírus, através da reaçäo de imunofluorescência indireta. Num total de 268 casos, obteve-se uma porcentagem de positividade de 14.93 por cento (40 amostras). A comparaçäo desta prova com o ensaio imunoenzimático (EIERA) e a eletroforese em gel de poliacrilamida(EGPA), revelou boa concordância entre os três ensaios, com valores de kappa entre 0.72 e 0.76.

A reaçäo de imunofluorescência indireta no diagnóstico de gastroenterites por rotavírus / The indirect immunofluorescence as say for the diagnosis of human rotavirus gastroenteritis

Utilizando a cepa de rotavírus SA-11, cultivada em células MA-104 e purificadas em gradiente de CsCl, soros antirotavírus foram preparados em cobaias para uso no diagnóstico de gastroenterites por rotavírus, através da reaçäo de imunofluorescência indireta. Do total de 268 casos estudados, analisados por imunofluorescência indireta, feita em microplacas com culturas de células MA-104, obteve-se uma porcentagem de positividade de 14,93% (40 amostras). A comparaçäo desta prova com o ensaio imunoenzimático (EIERA) e a eletroforese em gel de poliacrilamida (EGPA), revelou boa concordância entre os três ensaios, com valores para kappa entre 0,72 e 0,83, significativamente maior que a concordância obtida ao acaso.