While enteroviruses, including poliovirus, are conventionally released upon cell lysis, recent studies show that phosphatidylserine-enriched infectious extracellular vesicles (IEVs) shed by infected cells can transport clusters of enteroviruses from cell to cell, resulting in increased infectivity. Combining structural and biochemical analyses, we focused on IEVs shed from poliovirus-infected cells, a classical prototype for studying enteroviruses. Transmission cryo-electron microscopy, cryo-electron tomography and computational reconstruction, present the first three-dimensional structures of well-preserved IEVs and purified exosomes. We observed that single-membraned IEVs present a wide size range in diameter. Clusters of virions can be either densely packed within a protein-coated irregularly shaped IEV, or concentrated at one or both ends of an IEV, forming a polar structure. In addition to virions, IEVs often contain internal vesicles, “ramen-noodle”-like structures with strong density, and partially assembled virion-like structures. Viral replication complex components, including viral proteins polymerase 3D, 3CD, 3A, 3AB, 2BC, 2C and (+) and (-) stranded RNAs were detected in IEVs. Furthermore, (-) stranded RNA templates are protected by the IEVs, not packed in viral capsids. The transported viral replication components (viral proteins and RNAs) and virions within IEVs initiate a stronger and faster viral replication in recipient cells than free virions. Both cryo-electron tomographic and mass spectrometry data also showed that virions and “ramen-noodle”-like structures were also observed in purified CD9 positive exosomes from poliovirus-infected cells. Viral protein 3AB, detected on the membrane of IEVs, can invaginate membranous structures to engulf large proteins into a closed lumen. Our study demonstrates that IEVs can transport viral replication complex components to initiate a rapid onset of viral replication, as part of a novel viral transmission mechanism. Viral protein 3AB may contribute to forming IEVs throughout the infection. / 2019-06-12T00:00:00Z

Discovered in 1969 in California, enterovirus 71 (EV-71) is a serious cause of disease in young children. It is one of the major causative agents of hand, food, and mouth disease (HFMD), and can produce neurological complications, such as meningitis, encephalitis, and an acute flaccid paralysis. For serious cases, the fatality rate can be up to 26%, almost exclusively in young children. While the virus was initially discovered in the United States, it was soon detected in the Eastern hemisphere, causing outbreaks in Europe and Asia. The largest outbreak occurred in Taiwan in 2008, with approximately 490,000 cases and 128 fatalities. However, despite the seriousness of EV-71, there are currently no approved antiviral treatments. Physicians rely on supportive care and the off-label use of a purified antibody mixture, intravenous immunoglobulin, for treatment. Part of the difficulty in developing antivirals for EV-71 is a lack of drug testing in animal models. Animal testing is a crucial step in drug development, determining which compounds will progress to clinical trials in humans. However, viruses that cause disease in humans do not necessarily cause disease or the same type of disease in animals. As such, viruses often need to be adapted before they can cause disease in their animal hosts. Adaption isn’t always successful and can result in a virus that produces disease that is unlike that seen in humans. Furthermore, some animal models can produce disease only under a strict set of conditions, such as newborn mice. Sometimes these animal model conditions may be impractical for testing potential treatments. At the Institute for Antiviral Research (IAR), we developed an animal model for EV-71 in four-week-old AG129 mice. AG129 mice lack the alpha, beta, and gamma interferon receptors, making them immunocompromised. Being immunocompromised, these mice are more susceptible to infection, including infection from human viruses. In our model, EV-71 infection produces neurological signs, including a rear-limb paralysis (similar to the paralysis seen children with EV-71). The virus is also lethal in these animals, which provides an observable and consistent baseline for evaluating potential drugs. We assessed twenty-four potential treatments in our EV-71 model. Two compounds, STF434 and STF1019, provided 30% and 87% protection against mortality. Intravenous immunoglobulin was also examined and found to be about 50% protective against mortality, depending on the dose and time of administration. Intravenous immunoglobulin also reduced inflammatory modulators (cytokines) in the brain and spinal cord. We consider this to be highly relevant, given that inflammation is a serious component of EV-71 infection.

No description available.

Background: Enterovirus A71 is one of the causative agents of hand, foot, and mouth disease, which is usually a self-limiting disease. Complications of enterovirus infection are also very rare. However, when such complications occur, they can lead to serious neurological diseases or even death. Case presentation: In this report, we describe a case of enterovirus A71-associated acute flaccid paralysis in a 13-month-old Caucasian girl that was managed in our hospital. The patient presented with sudden onset of left arm paresis that could not be attributed to any other cause. Establishing a diagnosis was furthermore complicated by negative virological investigations of cerebrospinal fluid and non-pathological radiological findings. A polymerase chain reaction test of the child’s stool sample however tested positive for enterovirus and sequencing results revealed the presence of enterovirus A71. A previous history of febrile gastroenteritis just before the paresis started also supported the suspected diagnosis of enterovirus-associated acute flaccid paralysis. Following this, the child was treated with intravenous immunoglobulin over 5 days and a remarkable improvement was observed in the child’s paresis. Conclusion: This case report describes a possible complication of enterovirus A71 infection in a child. It also highlights the prolonged detection of enterovirus in the child’s stool sample as compared with cerebrospinal fluid weeks after the primary infection occurred. Finally, it shows the need for increased clinical and diagnostic awareness especially in the management of sudden and unknown causes of paresis or paralysis in children.

Lodos de esgoto são resíduos do tratamento do esgoto doméstico, considerados ricos em macronutrientes e matéria orgânica, podendo também apresentar contaminação por substâncias químicas e patógenos. Sua utilização na agricultura é uma das alternativas interessantes para sua disposição final. Entretanto, a presença de contaminantes, dentre eles microrganismos patogênicos podem limitar e orientar sua aplicação. Os vírus entéricos, dentre os quais se inclui o gênero Enterovirus, são potenciais contaminantes microbianos presentes no lodo de esgoto. Esses organismos são capazes de sobreviver por meses em águas e solos e sua presença no ambiente pode trazer prejuízos à saúde da população exposta aos mesmos. O objetivo do presente estudo foi de analisar a ocorrência de Enterovirus em amostras de lodo de esgoto de seis diferentes ETEs do Estado de São Paulo, avaliar o desempenho do método analítico para a detecção desses organismos e verificar se as concentrações médias obtidas atendem à legislação. Foram coletadas um total de 35 amostras no período de fevereiro de 2009 a dezembro de 2009. As análises dos Enterovirus foram realizadas pelo ensaio de plaqueamento em cultura de células RD segundo método EPA /625/R-092/013. Os resultados obtidos foram que Enterovirus estiveram presentes em 83 por cento das amostras analisadas, com concentrações que variaram de não detectado (ND) a 12,50 UFP/gST. As taxas de recuperação obtidas variaram de 0,20 por cento a 68,50 por cento . Conclui-se que das amostras analisadas 31 por cento atendem o estabelecido pela Resolução CONAMA 375/2006 / Sewage Sludge is a waste generated from wastewater treatment and is considered besides rich in macronutrients and organic matter, contaminated with pathogen and chemical substances. The usage of sludge in agriculture has been considered an interesting option. Nevertheless, the presence of contaminates such as pathogenic organisms can lead to usage limitations. Enteric viruses in wich are included enterovirus genera, a potential sludge contaminants. These organisms are able to survive for months in water and soil and their presence can bring public health concerns. The aim of this study was to analyse the occurrence of Enterovirus in samples of sewage sludge from six sewage plant treatment located in São Paulo State, to assess the method performance (recovery rate) in detecting these organisms and to verify the results obtained meet the standard established by the law. A total of 35 samples were collected spanning February to December 2009. The analyses were carried out according to method EPA/625/R-092/013. Enterovirus were present in 83 per cent of samples examined with concentration varied from not detected (ND) to 12.5 PFU/gTS. The recovery rate varied from 0,2 per cent to 68,50¨ per cent . This study showed that 31 per cent of analyzed samples met the standard established by Resolução CONAMA 375/2006

Lodos de esgoto são resíduos do tratamento do esgoto doméstico, considerados ricos em macronutrientes e matéria orgânica, podendo também apresentar contaminação por substâncias químicas e patógenos. Sua utilização na agricultura é uma das alternativas interessantes para sua disposição final. Entretanto, a presença de contaminantes, dentre eles microrganismos patogênicos podem limitar e orientar sua aplicação. Os vírus entéricos, dentre os quais se inclui o gênero Enterovirus, são potenciais contaminantes microbianos presentes no lodo de esgoto. Esses organismos são capazes de sobreviver por meses em águas e solos e sua presença no ambiente pode trazer prejuízos à saúde da população exposta aos mesmos. O objetivo do presente estudo foi de analisar a ocorrência de Enterovirus em amostras de lodo de esgoto de seis diferentes ETEs do Estado de São Paulo, avaliar o desempenho do método analítico para a detecção desses organismos e verificar se as concentrações médias obtidas atendem à legislação. Foram coletadas um total de 35 amostras no período de fevereiro de 2009 a dezembro de 2009. As análises dos Enterovirus foram realizadas pelo ensaio de plaqueamento em cultura de células RD segundo método EPA /625/R-092/013. Os resultados obtidos foram que Enterovirus estiveram presentes em 83 por cento das amostras analisadas, com concentrações que variaram de não detectado (ND) a 12,50 UFP/gST. As taxas de recuperação obtidas variaram de 0,20 por cento a 68,50 por cento . Conclui-se que das amostras analisadas 31 por cento atendem o estabelecido pela Resolução CONAMA 375/2006 / Sewage Sludge is a waste generated from wastewater treatment and is considered besides rich in macronutrients and organic matter, contaminated with pathogen and chemical substances. The usage of sludge in agriculture has been considered an interesting option. Nevertheless, the presence of contaminates such as pathogenic organisms can lead to usage limitations. Enteric viruses in wich are included enterovirus genera, a potential sludge contaminants. These organisms are able to survive for months in water and soil and their presence can bring public health concerns. The aim of this study was to analyse the occurrence of Enterovirus in samples of sewage sludge from six sewage plant treatment located in São Paulo State, to assess the method performance (recovery rate) in detecting these organisms and to verify the results obtained meet the standard established by the law. A total of 35 samples were collected spanning February to December 2009. The analyses were carried out according to method EPA/625/R-092/013. Enterovirus were present in 83 per cent of samples examined with concentration varied from not detected (ND) to 12.5 PFU/gTS. The recovery rate varied from 0,2 per cent to 68,50¨ per cent . This study showed that 31 per cent of analyzed samples met the standard established by Resolução CONAMA 375/2006

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 126-147). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Nucleic acid hybridization (gene probe) and polymerase chain reaction (PCR) techniques have been used to detect viral nucleic acid in water. However, gene probe and PCR may not distinguish between infectious and noninfectious viruses. This study evaluated the ability of gene probe to detect viable poliovirus 1 (polio 1), from sterile and nonsterile groundwater, and the ability of PCR to detect infectious human immunodeficiency virus (HIV-1) from tap and wastewater. The plaque forming (BGM cells), and the tissue culture infectious dose fifty (TCID₅₀) (PLC/PRF/5 cells) procedures were used to detect infectious polio 1 and HIV-1, respectively. Detection of polio 1 by gene probe and cell culture was similar in nonsterile water and in filter sterilized water, but not in autoclaved water. These results suggest that in some natural waters, detection of polio 1 by gene probe may correlate to detection by cell culture procedures. Although detection of infectious HIV-1 by cell culture decreased gradually, until no virus could be found, detection by PCR remained positive throughout the study. Therefore, it was concluded that the use of PCR to assess the risk associated to the presence of HIV-1 in polluted waters, may not be adequate. The enteric adenovirus types 40 (Ead 40) and 41 (Ead 41) are considered the second most important cause of viral gastroenteritis in children, but their role as waterborne pathogens is uncertain. This study compared the survival of Ead 40 and Ead 41 with polio 1, and hepatitis A virus (HAV) in different types of water. The Enteric adenoviruses survived longer in tap and sea water than either polio 1 or HAV, but only slightly better in wastewater. These results suggest that the enteric adenoviruses may survive for prolonged periods in water, representing a potential route of transmission. This study evaluated also the concentration of Ead 40 by the filter adsorption-elution method. With negatively-charged filters, recovery efficiencies of 22, 36, and 38% were obtained from secondary sewage, tap and sea water, respectively. Using electropositive filters, Ead 40 was recovered from tap water with an efficiency of 26.5%. These results show that Ead 40 can be concentrated, from water, with an efficiency comparable to that of other enteric viruses.

The Centers for Disease Control and Prevention estimated 23,000,000 cases of viral gastroenteritis caused by Norovirus in 2000, 40% of which were transmitted by food including: a variety of fresh produce, cake, deli meats, fruit salad, cheeses and ice. (CDC, 2003). An estimated 83,391 cases of Hepatitis A virus was reported in 2000, of which 5% was attributed to foodborne transmission (CDC, 2003). These figures underscore an urgent need for a method that can isolate virus from a variety of food matrices. The aim of this study was to develop an overall assessment of the inhibitory effects of a variety of food matrices on Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Additionally, to compare a sequence specific hybridization probe amplification format to a non sequence specific SYBR Green format using the Roche LightCycler. The secondary aim was to evaluate the effectiveness of a food virus concentration and isolation protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa. Three food specimens consisting of prepackaged smoked ham, fresh cilantro, and Thompson's green grapes were seeded with three dilutions of poliovirus 3 (Sabin strain). A viral concentration procedure under development at the Florida Department of Health Bureau of Laboratories, Tampa was used to isolate the virus. Real Time RT-PCR was carried out on the Roche LightCycler in SYBR Green and Hybridization probe formats. Spiking the virus-negative samples of each matrix with a dilution series of poliovirus 3 created post flocculation spikes. This post-flocculation dilution series amplification allowed a standard curve to be created unique to each food matrix. The flocculation and concentrations specimens were then amplified and the standard curves from the post-flocculation seed were used to calculate the loss associated with the concentration procedure. This study reports significant differences (p<0.05) in recovery detected between the various matrices, and Real Time RT-PCR formats. The concentration protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa, demonstrates a 12-78% recovery of seeded virus in a simulated “real world” virus contamination event among the various matrices.