Nipah Virus in the Fruit Bat Pteropus vampyrus in Sumatera, Indonesia

Nipah virus causes periodic livestock and human disease with high case fatality rate, and consequent major economic, social and psychological impacts. Fruit bats of the genus Pteropus are the natural reservoir. In this study, we used real time PCR to screen the saliva and urine of P. vampyrus from North Sumatera for Nipah virus genome. A conventional reverse transcriptase (RT-PCR) assay was used on provisionally positive samples to corroborate findings. This is the first report of Nipah virus detection in P. vampyrus in Sumatera, Indonesia.

Phage M13Ko7 Detection With Biosensor Based On Imaging Ellipsometry And Afm Microscopic Confirmation

A rapid detection and identification of pathogens is important for minimizing transfer and spread of disease. A label-free and multiplex biosensor based on imaging ellipsometry (BIE) had been developed for the detection of phage M13KO7. The surface of silicon wafer is modified with aldehyde, and proteins can be patterned homogeneously and simultaneously on the surface of silicon wafer in an array format by a microfluidic system. Avidin is immobilized on the surface for biotin-anti-M13 immobilization by means of interaction between avidin and biotin, which will serve as ligand against phage M13KO7. Phages M13KO7 are specifically captured by the ligand when phage M13KO7 solution passes over the surface, resulting in a significant increase of mass surface concentration of the anti-M13 binding phage M13KO7 layer, which could be detected by imaging ellipsometry with a sensitivity of 10(9) pfu/ml. Moreover, atomic force microscopy is also used to confirm the fact that phage M13KO7 has been directly captured by ligands on the surface. It indicates that BIE is competent for direct detection of phage M13KO7 and has potential in the field of virus detection. (C) 2008 Elsevier B.V. All rights reserved.

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

Propidium monoazide (PMA) and ethidium bromide monoazide(EMA) improve DNA array and high-throughput sequencing ofporcine reproductive and respiratory syndrome virus identification

Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses.

One-step reverse transcriptase polymerase chain reaction for the diagnosis of respiratory syncytial virus in children

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory tract infections in infants and children. Rapid diagnosis is required to permit appropriate care and treatment and to avoid unnecessary antibiotic use. Reverse transcriptase (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been considered important tools for virus detection due to their high sensitivity and specificity. In order to maximize use-simplicity and minimize the risk of sample cross-contamination inherent in two-step techniques, a RT-PCR method using only a single tube to detect HRSV in clinical samples was developed. Nasopharyngeal aspirates from 226 patients with acute respiratory illness, ranging from infants to 5 years old, were collected at the University Hospital of the University of Sao Paulo (HU-USP), and tested using IFA, one-step RT-PCR, and semi-nested RT-PCR. One hundred and two (45.1%) samples were positive by at least one of the three methods, and 75 (33.2%) were positive by all methods: 92 (40.7%) were positive by one-step RT-PCR, 84 (37.2%) by IFA, and 96 (42.5%) by the semi-nested RT-PCR technique. One-step RT-PCR was shown to be fast, sensitive, and specific for RSV diagnosis, without the added inconvenience and risk of false positive results associated with semi-nested PCR. The combined use of these two methods enhances HRSV detection. (C) 2007 Elsevier B.V. All rights reserved.

Replication of classical infectious bursal disease virus in the chicken embryo related cell line

Infectious bursal disease (IBD) is an acute, highly contagious viral disease. The diagnosis of IBD depends on time-consuming and costly procedures, like virus isolation on chick embryos and histopathological examination, A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunoperoxidase and reverse transcription polymerase chain reaction (RT-PCR) were applied in this study to detect classical IBD virus (IBDV) after three blind passages of the Lukert strain on chicken embryo related (CER) cell monolayer after different periods of infection: 6, 12, 24 and 48 h, Cytophatic effects were most evident 12 h post-infection (p.i.) but were observed at 6 h p.i. The maximum discrimination between IBDV-infected and uninfected cell suspensions obtained by the use of DAS-ELISA for virus detection corresponded to 0.597+/-0.02 and 0.010+/-0.01 after 12h p.i., respectively. The RT-PCR was performed using the set of primers A3.1 and A3.2 to amplify the VP2 region of the IBDV genome, This molecular technique demonstrated that from 6 h p.i., it was possible to detect the viral RNA. The results show that the CER cell line can be used for classical IBDV propagation, confirmed by the DAS-ELISA, immunoperoxidase and RT-PCR assay.

Circulation of Different Lineages of Dengue Virus 2, Genotype American/Asian in Brazil: Dynamics and Molecular and Phylogenetic Characterization

The American/Asian genotype of Dengue virus type 2 (DENV-2) was introduced into the Americas in the 80′s. Although there is no data showing when this genotype was first introduced into Brazil, it was first detected in Brazil in 1990. After which the virus spread throughout the country and major epidemics occurred in 1998, 2007/08 and 2010. In this study we sequenced 12 DENV-2 genomes obtained from serum samples of patients with dengue fever residing in São José do Rio Preto, São Paulo (SJRP/SP), Brazil, in 2008. The whole open reading frame or envelope sequences were used to perform phylogenetic, phylogeographic and evolutionary analyses. Isolates from SJRP/SP were grouped within one lineage (BR3) close to isolates from Rio de Janeiro, Brazil. Isolates from SJRP were probably introduced there at least in 2007, prior to its detection in the 2008 outbreak. DENV-2 circulation in Brazil is characterized by the introduction, displacement and circulation of three well-defined lineages in different times, most probably from the Caribbean. Thirty-seven unique amino acid substitutions were observed among the lineages, including seven amino acid differences in domains I to III of the envelope protein. Moreover, we dated here, for the first time, the introduction of American/Asian genotype into Brazil (lineage BR1) to 1988/89, followed by the introduction of lineages BR2 (1998-2000) and BR3 (2003-05). Our results show a delay between the introduction and detection of DENV-2 lineages in Brazil, reinforcing the importance and need for surveillance programs to detect and trace the evolution of these viruses. Additionally, Brazilian DENV-2 differed in genetic diversity, date of introduction and geographic origin and distribution in Brazil, and these are important factors for the evolution, dynamics and control of dengue. © 2013 Drumond et al.

Caracterização biofísica e produção de antissoro policlonal contra a capa proteica recombinante do Southem bean mosaic virus

Pós-graduação em Genética - IBILCE

High Rates of Detection of Respiratory Viruses in Tonsillar Tissues from Children with Chronic Adenotonsillar Disease

Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases.

Virological and pathological findings in Bluetongue virus serotype 8 infected sheep

Twenty-seven sheep of the four most common Swiss breeds and the English breed Poll Dorset were experimentally infected with a northern European field strain of bluetongue virus serotype 8 (BTV-8). Animals of all breeds developed clinical signs, viremia and pathological lesions, demonstrating that BTV-8 is fully capable of replicating and inducing bluetongue disease (BT) in the investigated sheep. Necropsy performed between 10 and 16 days post-infectionem (d.p.i.) revealed BT-typical hemorrhages, effusions, edema, erosions and activation of lymphatic tissues. Hemorrhages on the base of the Arteria pulmonalis and the left Musculus papillaris subauricularis were frequently present. Histology confirmed the macroscopical findings. Using a score system, clinical manifestation and pathology were found to be significantly related. Furthermore, clinical signs and fever were shown to be indicative for the concurrent presence of high amounts of viral ribonucleic acid (RNA) in blood. Spleen, lung, lymph nodes and tonsils from all animals were analyzed regarding viral RNA loads and infectivity using real-time reverse transcriptase PCR (rRT-PCR) and virus isolation in cell culture, respectively. The highest amount of viral RNA was detected in spleen and lung and rRT-PCR revealed to be a more sensitive method for virus detection compared to virus isolation. A long-term follow-up was performed with three sheep showing that BTV-8 viral RNA in blood was present up to 133 d.p.i. and in certain tissues even on 151 d.p.i. No significant breed-related differences were observed concerning clinicopathological picture and viremia, and the Swiss sheep were as susceptible to BTV-8 infection as Poll Dorset sheep, demonstrating a remarkably high virulence of BTV-8 for indigenous sheep breeds.

Applications of a sugar-based surveillance system to track arboviruses in wild mosquito populations

Effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. Traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. A system was recently developed that involves collecting mosquitoes in CO2-baited traps, where the insects expectorate virus on sugar-baited nucleic acid preservation cards. The cards are then submitted for virus detection using molecular assays. We report the application of this system for detecting flaviviruses and alphaviruses in wild mosquito populations in northern Australia. This study was the first to employ nonpowered passive box traps (PBTs) that were designed to house cards baited with honey as the sugar source. Overall, 20/144 (13.9%) of PBTs from different weeks contained at least one virus-positive card. West Nile virus Kunjin subtype (WNVKUN), Ross River virus (RRV), and Barmah Forest virus (BFV) were detected, being identified in 13/20, 5/20, and 2/20 of positive PBTs, respectively. Importantly, sentinel chickens deployed to detect flavivirus activity did not seroconvert at two Northern Territory sites where four PBTs yielded WNVKUN. Sufficient WNVKUN and RRV RNA was expectorated onto some of the honey-soaked cards to provide a template for gene sequencing, enhancing the utility of the sugar-bait surveillance system for investigating the ecology, emergence, and movement of arboviruses. © 2014, Mary Ann Liebert, Inc.

Otimização da RT-PCR Multiplex para detecção de vírus dengue em amostras de Aedes aegypti infectados artificialmente

A dengue é a mais importante doença viral transmitida por mosquitos, no que diz respeito à morbidade e mortalidade, que afeta os seres humanos. Este vírus é transmitido pelos vetores Aedes albopictus e Aedes aegypti, este último é o principal vetor nas Américas. O controle da doença se baseia na vigilância laboratorial e vigilância entomológica. A vigilância laboratorial visa aprimorar a capacidade do diagnóstico, detectando precocemente a circulação viral e monitorando os sorotipos circulantes. Dentro deste tipo de vigilância, a RT-PCR é um método bastante usado no diagnóstico da doença em humanos e mosquitos, porém, a má conservação do material pode comprometer a integridade do RNA e trazer resultados falso-negativos. O desenvolvimento de melhores métodos de vigilância do vírus dengue (DENV) em mosquitos é de grande valor para os programas de controle. Desta maneira, o presente projeto visou otimizar a técnica de RT-PCR Multiplex para detecção de DENV em amostras de Ae. aegypti infectadas artificialmente pelo vírus. Primers que amplificam uma região de 80 pb do gene rpL8 de mosquito foram desenhados no site Primer3 e avaliados na ferramenta online Multiple Primer Analyzer, junto com primers que amplificam os sorotipos DENV. Não houve competição de primers e foi observado bandas distintas no gel de agarose. Foi avaliado o efeito de diferentes formas de preservação do material genético das amostras (RNAlater®, freezer -80°C e nitrogênio líquido) por 7 dias, onde não houve diferenças significativas em relação à integridade do RNA. O efeito de diferentes formas de extração de RNA (Kit da QIAGEN® , TRIzol® e Chomczymski-Sacchi) também foi avaliado e o método ChomczymskiSacchi obteve o melhor desempenho. A otimização desta técnica permitirá uma maior confiabilidade nos resultados, já que além da detecção dos sorotipos, haverá uma confirmação da qualidade do RNA, aprimorando a capacidade do diagnóstico e auxiliando a prevenção e controle da transmissão da dengue.

Comparação entre dois métodos de detecção de DNA de papilomavírus humano em carcinoma epidermoide de lábio

Introdução: Recentemente o papilomavírus humano (HPV) tem sido associado à carcinogênese oral. A metodologia empregada na detecção do vírus é uma das maiores causas observadas da grande variabilidade nas taxas de detecção do HPV. Objetivo: Este estudo comparou a sensibilidade de detecção do DNA do HPV em casos de carcinoma epidermoide de lábio utilizando a amplificação do DNA viral por reação em cadeia da polimerase (PCR) ou nPCR. Material e método: Foram utilizadas 33 amostras provenientes de casos de carcinoma epidermoide de lábio. Para as extrações do DNA utilizou-se o sistema QIAamp DNA Mini Kit. Como controle interno utilizou-se o gene da b-globina. Das 33 amostras iniciais, 30 foram positivas para o gene b-globina, sendo utilizadas para detectar o DNA viral. Comparou-se a amplificação do DNA viral pelos métodos da PCR com os oligonucleotídeos MY09/MY11 e nPCR, empregando-se os pares de oligonucleotídeos iniciadores MY09/MY11 e, na segunda etapa, o par GP5+/GP6+. O controle positivo para a presença do DNA do HPV utilizado foi a linhagem de células HeLa e, como controle negativo, a mistura de amplificação sem DNA. A análise dos produtos de PCR e nPCR para HPV foi realizada por eletroforese em gel de poliacrilamida a 8%. Resultados: Utilizando-se o método da PCR, a amplificação do DNA do HPV foi constatada em dois casos. Com a nPCR foi verificada presença de DNA viral em 13 das 30 amostras. Conclusão: Com a utilização da nPCR, a detecção do HPV nos casos estudados aumentou mais de seis vezes.

Vírus da leucemia felina: análise da classificação da infecção, das técnicas de diagnóstico e da eficácia da vacinação com o emprego de técnicas sensíveis de detecção viral

O Vírus da leucemia felina (FeLV) pertence à família Retroviridae, gênero Gammaretrovirus. Diferentemente de outras retroviroses, uma parcela dos gatos jovens e adultos exposta ao FeLV não apresenta antigenemia/viremia, de acordo com as técnicas convencionais de detecção viral, como isolamento em cultivo celular, imunofluorescência direta e ELISA. O emprego de técnicas de maior sensibilidade para detecção e quantificação viral, como o PCR quantitativo, permitiu a identificação de animais positivos para a presença de DNA proviral e RNA na ausência de antigenemia/viremia e, com isso, um refinamento da análise das diferentes evoluções da infecção. Assim, reclassificou-se a patogenia do FeLV em 4 categorias: infecção abortiva, regressiva, latente e progressiva. Foi possível também detectar DNA proviral e RNA em animais considerados imunes ao FeLV após vacinação. Diante disso, os objetivos desta revisão de literatura foram demonstrar as implicações da utilização de técnicas sensíveis de detecção viral na interpretação e classificação da infecção do FeLV e rever as técnicas de detecção do vírus para fins de diagnóstico. Além disso, apresentar os resultados referentes à eficácia da vacinação contra o FeLV com a utilização dessas técnicas.

Occurrence, distribution, and relative incidence of five viruses infecting cucurbits in the state of Sao Paulo, Brazil

Cucurbits species grown in 38 of 40 agricultural regions in the state of Sao Paulo, Brazil, were surveyed for the relative incidence of Cucumber mosaic virus (CMV), Papaya ringspot virus-type W (PRSV-W), Watermelon mosaic virus-2 (WMV-2), Zucchini lethal chlorosis virus (ZLCV), and Zucchini yellow mosaic virus (ZYMV) during May 1997 and June 1999. Samples from 621 plants, representing eight cultivated species, six wild species, and one commercial hybrid (Cucurbita moschata x C. maxima), were analyzed by plate trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA). PRSV-W and ZYMV were the most frequently found viruses, accounting for 49.1 and 24.8%, respectively, of 605 samples tested. ZLCV, CMV, and WMV-2 were detected in 7.8, 6.0, and 4.5% of 612, 497, and 423 samples tested, respectively. Double infection was found in 97 samples, and triple infection was found in 10 samples. Quadruple infection was detected in one C. pepo sample. Plants that were symptomatic but negative by PTA-ELISA might be due to abiotic agents, infection by virus for which antiserum was not available, such as Squash mosaic virus, or infection with an as yet uncharacterized virus.