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Viruses are masters at using cellular pathways to aid their replication. Cryo-electron tomography of poliovirus-infected cells revealed how it utilizes macroautophagy to its advantage. Assembly of these non-enveloped virions takes place directly on membranes and requires PIK3C3/VPS34 activity to be completed, whereas the canonical autophagy inducer ULK1 restricts virus assembly. The tomograms further revealed that enterovirus-induced autophagy is selective for RNA-loaded virions, which may help ensure maximum infectivity of the virus-laden vesicles released through secretory autophagy., Published online: 05 Dec 2022

There is a growing interest in echovirus 30 (E30), an enterovirus responsible for neurological disease and hospitalization. There are multiple studies of outbreaks, but few that study the epidemiology over long periods of time. Our study aims to describe the clinical, epidemiological and microbiological characteristics of a series of E30 infections detected over 26 years. Data were retrospectively collected from a database of all enterovirus infections identified in our laboratory. They were detected by viral isolation or nucleic acid detection in patients presenting with respiratory or neurological infections, rash, sepsis-like syndrome, or gastroenteritis. Enterovirus genotyping was performed by amplification of the VP1 gene using RT-nested PCR, followed by sequencing and BLAST analysis. Of the 2402 enterovirus infections detected, 1619 were linked to at least one genotype and 173 were caused by E30. Clinical information was available for 158 (91.3%) patients. E30 was associated with neurological infection in 107 (67.8%) cases and it was detected almost every year. Phylogenetic analysis was performed with 67 sequences. We observed that E30 strains circulating in Catalonia from 1996 to 2016 belong to two lineages (E and F), although the majority cluster was in F. In 2018, lineage I emerged as the dominant lineage.

INTRODUCCIÓ. Les infeccions neonatals son una patologia molt prevalent i representen una de les principals causes de mortalitat durant aquest període de la vida. Gran part dels nadons que ingressen durant el primer mes de vida amb sospita de sèpsia bacteriana (SB) són diagnosticats d'infeccions produïdes per virus. Els virus més implicats en la infecció neonatal són els enterovirus (EV) amb una incidència de 7 casos per cada 1000 nadons. L'objectiu d'aquest estudi és demostrar la importància dels EV com a agents etiològics de la infecció neonatal per les seves diferències pel que fa al maneig clínic i les mesures terapèutiques a seguir. MATERIAL I MÈTODES. El present treball es un estudi retrospectiu amb recollida prospectiva de les dades. Es van incloure un total de 332 nadons d'edat gestacional > 34 SG atesos a la Unitat de Neonatologia de l'Hospital de la Santa Creu i Sant Pau entre gener de l'any 2002 fins a desembre del 2017 i amb el diagnòstic inicial de sèpsia neonatal (SN). RESULTATS. El percentatge d'infeccions per EV va ser superior al de SB amb un 34,6% i 32,5% respectivament del total de nadons estudiats. Els nadons amb infecció per EV presenten menor patologia materna a excepció de la malaltia hipertensiva de l'embaràs (p <0,001). Es tracta de nadons sans amb més edat gestacional, majors puntuacions en el test d'Apgar i major pes al naixement (p <0,001). En la meitat dels casos hi ha antecedents d'ambient epidèmic familiar (p <0,001). La febre es va presentar en el 72% dels nadons amb infecció per EV i la meningitis va ser la forma clínica més freqüent afectant el 61,7% dels casos d'infecció per EV. Les característiques del LCR dels pacients amb meningitis van ser similars en ambdós grups i només van existir diferències significatives en la concentració de proteïnes, superior en el grup de SB (p> 0,001). Les formes greus de la malaltia van ser poc freqüents (1,7%). El 51,4% dels pacients amb infecció per EV va ser sotmès a tractament antibiòtic (ATB). L, INTRODUCCIÓN. Las infecciones neonatales constituyen una de las patologías más prevalentes y representan una de las principales causas de mortalidad durante este periodo de la vida. Gran parte de los recién nacidos (RN) que ingresan durante el primer mes de vida con sospecha de sepsis bacteriana (SB) son diagnosticados de infecciones producidas por virus. Los virus mayormente implicados en la infección neonatal son los enterovirus (EV) con una incidencia de 7 casos por cada 1000 RN. El objetivo de este estudio es demostrar la importancia de los EV como agentes etiológicos en la infección neonatal por sus diferencias en cuanto al manejo clínico y las medidas terapéuticas a seguir. MATERIAL Y METODOS. El presente trabajo consiste en un estudio retrospectivo con recogida prospectiva de los datos. Se incluyeron un total de 332 RN de edad gestacional > 34 semanas gestacionales (SG) atendidos en la Unidad de Neonatología del Hospital de la Santa Creu i Sant Pau entre enero del 2002 hasta diciembre del 2017 y con el diagnóstico inicial de sepsis neonatal (SN). RESULTADOS. El porcentaje de infecciones por EV fue superior al de SB con un 34,6% y 32,5% respectivamente del total de RN estudiados. Los RN con infección por EV presentan menor patología materna a excepción de la enfermedad hipertensiva del embarazo (p<0,001), son RN sanos con mayor edad gestacional, mayores puntuaciones de Apgar y mayor peso al nacimiento (p<0,001) y en la mitad de los casos existen antecedentes de ambiente epidémico familiar (p<0,001). La fiebre se presenta en el 72% de los RN con infección por EV y la meningitis es la forma clínica más frecuente afectando al 61,7% de los casos. Las características del líquido cefalorraquídeo (LCR) de los pacientes con meningitis fueron similares en ambos grupos y solo encontramos diferencias significativas en la concentración de proteínas, superior en el grupo de SB (p>0,001). Las formas graves de la enfermedad fueron poco frecuentes (1,7%). El 51,4% de los paci, INTRODUCTION. Neonatal infections are one of the most prevalent pathologies and represent one of the main causes of mortality during this period of life. Most of the newborns who are admitted during the first month of life with suspected Bacterial Sepsis (BS) are diagnosed with infections caused by viruses. The viruses most involved in neonatal infection are enteroviruses (EV), with an incidence of 7 cases per 1000 newborns. The objective of this study is to demonstrate the importance of EV as etiological agents in neonatal infection due to their differences in terms of clinical management and the therapeutic measures to be followed. MATERIAL AND METHODS. The present work consists of a retrospective study with prospective data collection. A total of 332 NBs of gestational age> 34 SG attended in the Neonatology Unit of the Hospital de la Santa Creu i Sant Pau between January 2002 and December 2017 and with the initial diagnosis of Neonatal Sepsis (NS) were included. RESULTS. The percentage of infections by EV was higher than that of BS with 34,6 and 32,5% respectively of the total of newborns studied. Newborns with EV infection present less maternal pathology except for hypertensive disease of pregnancy (p <0.001). Newborns with EV infection present less maternal pathology except for hypertensive disease of pregnancy (p <0.001), they are healthy newborns with a higher gestational age, higher Apgar scores and higher birth weight (p <0.001) and in the middle of the cases there is a history of a family epidemic environment (p <0.001). Fever occurs in 72% of newborns with EV infection and meningitis is the most frequent clinical form, affecting at 61.7% of EV infection cases. The CSF characteristics of patients with meningitis were similar in both groups and we only found significant differences in protein concentration, higher in the BS group (p> 0.001). Severe forms of the disease were rare (1.7%). 51.4% of the patients with EV infection underwent ATB treatment. The de, Universitat Autònoma de Barcelona. Programa de Doctorat en Pediatria, Obstetrícia i Ginecologia

The capsid precursor P1 constitutes the N-terminal part of the enterovirus polyprotein. It is processed into VP0, VP3, and VP1 by the viral proteases, and VP0 is cleaved autocatalytically into VP4 and VP2. We observed that poliovirus VP0 is recognized by an antibody against a cellular autophagy protein, LC3A. The LC3A-like epitope overlapped the VP4/VP2 cleavage site. Individually expressed VP0-EGFP and P1 strongly colocalized with a marker of selective autophagy, p62/SQSTM1. To assess the role of capsid proteins in autophagy development we infected different cells with poliovirus or encapsidated polio replicon coding for only the replication proteins. We analyzed the processing of LC3B and p62/SQSTM1, markers of the initiation and completion of the autophagy pathway and investigated the association of the viral antigens with these autophagy proteins in infected cells. We observed cell-type-specific development of autophagy upon infection and found that only the virion signal strongly colocalized with p62/SQSTM1 early in infection. Collectively, our data suggest that activation of autophagy is not required for replication, and that capsid proteins contain determinants targeting them to p62/SQSTM1-dependent sequestration. Such a strategy may control the level of capsid proteins so that viral RNAs are not removed from the replication/translation pool prematurely.

Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as a replication inhibitor of enterovirus B and D species. Fluoxetine likely targets the nonstructural viral protein 2C, but detailed mode-of-action studies are missing because structural information on 2C of fluoxetine-sensitive enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral activity of fluoxetine is stereospecific concomitant with binding to recombinant 2C. (S)-Fluoxetine inhibits with a 5-fold lower 50% effective concentration (EC50) than racemic fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive enterovirus coxsackievirus B3 (CVB3) based upon a recently elucidated structure of a fluoxetine-insensitive enterovirus, we predicted stable binding of (S)-fluoxetine. Structure-guided mutations disrupted binding and rendered coxsackievirus B3 (CVB3) resistant to fluoxetine. The study provides new insights into the anti-enteroviral mode-of-action of fluoxetine. Importantly, using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely reducing side effects.

Enteroviruses are potentially linked to the emergence of Acute Flaccid Myelitis (AFM), a rare but very serious condition that affects the nervous system. AFM has been associated with coxsackievirus A16, enterovirus A71 (EVA71) and enterovirus D68 (EVD68). Little is known about host-pathogen interactions for these viruses, and whether immune responses may have a protective or immunopathological role in disease presentations. Towards addressing this issue, we used the Immune Epitope Database to assess the known inventory of B and T cell epitopes from enteroviruses, focusing on data related to human hosts. The extent of conservation in areas that are targets of B and T cell immune responses were examined. This analysis sheds light on regions of the enterovirus polypeptide that can be probed to induce a specific or cross-reactive B or T cell the immune response to enteroviruses, with a particular focus on coxsackievirus A16, EVA71 and EVD68. In addition, these analyses reveal the current gap-of-knowledge in the T and B cell immune responses that future studies should aim to address.

Актуальність. Актуальність нейроінфекцій обумовлена вираженою тяжкістю перебігу та високим відсотком летальності. Кількість серозних менінгітів збільшується щодня. Серозний менінгіт є одним із найбільш частих проявів ентеровірусної інфекції. Мета. Встановлення опорних клініко-лабораторних симптомів ентеровірусної етіології менінгіту. Матеріали та методи. Проаналізували результати динамічного клінічного спостереження й обстеження 70 пацієнтів із менінгітами. За результатами полімеразної ланцюгової реакції калу та/або ліквору у 33 хворих встановлено ентеровірусну етіологію захворювання, а у решти 37 етіологія залишилася нез’ясованою. Результати. З’ясували, що ентеровірусні менінгіти мають ряд анамнестичних, клінічних і лабораторних особливостей, таких як: до ентеровірусного пошкодження менінгеальних оболонок більш схильні діти перших десяти років життя; важливим епідфактором при цьому є відпочинок на морі; серед ранніх симптомів ентеровірусного менінгіту частіше реєструється блювання; для ентеровірусного менінгіту характерна полога температурна крива з ранньої нормалізацією температури тіла (на 3–5-й день у більшості пацієнтів); у цих пацієнтів переважно має місце змішаний плеоцитоз ліквору., Актуальность. Актуальность нейроинфекций обу­слов-лена выраженной тяжестью течения и высоким процентом летальности. Количество серозных менингитов увеличивается ежедневно. Серозный менингит является одним из наиболее частых проявлений энтеровирусной инфекции. Цель. Установление опорных клинико-лабораторных симптомов энтеро­вирусной этиологии менингита. Материалы и методы. Про­анализировали результаты динамического клинического наблюдения и обследования 70 пациентов с менингитами. По результатам полимеразной цепной реакции кала и/или ликвора у 33 больных установлена энтеровирусная этиология заболевания, а у остальных 37 этиология осталась неясной. Результаты. Выяснили, что энтеровирусные менингиты имеют ряд анамнестических, клинических и лабораторных особенностей, таких как: энтеровирусному повреждению менингеальных оболочек более подвержены дети первых десяти лет жизни; важным эпидфактором при этом является отдых на море; среди ранних симптомов энтеровирусного менингита чаще регистрируется рвота; для энтеровирусного менингита характерна пологая температурная кривая с ранней нормализацией температуры тела (к 3–5-му дню у большинства пациентов); у этих пациентов преимущественно имеет место смешанный плеоцитоз ликвора., Background. The relevance of neuroinfections is explained by the severity of its course and the high mortality rate. The number of serous meningitis increases every day. Serous meningitis is one of the most common manifestations of enterovirus infection. Therefore, the purpose of our study was to establish the main clinical and laboratory symptoms of enterovirus meningitis. Materials and methods. We have analyzed the results of dynamic clinical observation and examination of 70 patients with meningitis. According to the results of polymerase chain reaction of stool and/or cerebrospinal fluid, in 33 of them the disease was of enterovirus origin, while in the remaining 37 subjects the etiology remained unexplained. Results. We found that enterovirus meningitis has a number of anamnestic, clinical and laboratory features such as: the children of the first ten years of life are more likely to be affected with enterovirus lesions of the meningeal membranes; the important epidemiological factor is holiday at the seaside; vomiting is most often reported among the early symptoms of enterovirus meningitis; the disease is characterized by a flat temperature curve with early normalization of body temperature (by day 3–5 in the majority of the patients); patients predominantly have a mixed cerebrospinal fluid pleocytosis.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), Institute for Glycomics, Science, Environment, Engineering and Technology, Rotavirus is a clinically significant pathogen and is the causative agent of life-threatening gastroenteritis in many young humans and animals, leading to approximately 500,000 deaths annually, with 85% occurring in developing countries globally. The mature infectious rotavirus particle, also called triple-layered particles (TLPs), is formed by three concentric layers of protein that surround the viral genome composed of eleven segments of doublestranded RNA (dsRNA). One of the essential steps in the rotavirus infection is the viral recognition and the recruitment of the host cell surface receptors, followed by the entry of the virion into the host cell cytoplasm triggering the viral replication process. Therefore, an understanding of the mechanism associated with the initial viral-host cell interactions would undoubtedly impart a critical insight that may lead to the development of an effective therapeutic. The rotavirus cell entry process is a complicated process and is poorly understood. The outermost layer of the virion is composed of two major proteins, the glycoprotein VP7, and the spike protein VP4. The VP4 spike protein plays a significant role in receptor binding and cell penetration. Rotavirus infectivity is significantly enhanced by the proteolytic cleavage of VP4 into its functional fragments VP8* and VP5* that remain associated with the virion. The globular head of the spike VP8*, mediates initial rotavirus interactions with cell membrane glycoconjugates, including those containing sialic acid (Sia). Here, we report the first X-ray crystal structure of the rotavirus carbohydrate-recognizing protein VP8* from porcine strain CRW-8 in complex with the GM1bGc ganglioside (which is the N-glycolyl form of GM1b) (Chapter 3). Our structure not only provides the first critical insights into the molecular basis of the interaction between rotavirus VP8* and a larger ganglioside, but has also allowed us to map the nature of rotavirus interactions with other ganglio

Type 1 diabetes (T1D) is an autoimmune disease that destroys the bodies’ insulin producing beta-cells. The disease is understood to be triggered in genetically susceptible individuals by environmental factors. While the genetic side of the etiological model has to some degree been uncovered, there is no clear understanding of which environmental factors play a role in the disease process. Several hypotheses claim to explain the development of T1D, of which enteroviral infections show the most promise. According to this hypothesis high prevalence of enteroviral infections would also mean high incidence rates of T1D. This study focused on four Nordic countries (Denmark, Finland, Norway and Sweden) that as late as 2017 were found in the top 10 countries for incidence rate of childhood-onset T1D in the world. Incidence rates of T1D and prevalence of enteroviruses were mapped and geographically analyzed according to the principles of spatial epidemiology, after which correlation coefficients were calculated. In doing so the study tried to answer to which extent the prevalence of enteroviruses could explain the regional variations in T1D. For all countries no significant correlation was found, but increasing sample size, by grouping countries, showed considerably different outcomes with a small positive correlation in the case of Norway and Finland.

Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed. To detect the presence of very low copies of the virus genome in tissue samples from T1D patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family. With these probes enteroviral RNA was detected with high sensitivity and specificity in infected cells and tissues, including in FFPE pancreas sections from patients with T1D. Detection was not impeded by variations in sample processing and storage thereby overcoming the potential limitations of fragmented RNA. Co-staining of small RNA probes in parallel with classical immunstaining enabled virus detection in a cell-specific manner and more sensitively than by viral protein.

Objective of the study was determination of enterovirus genom presence in the blood serum of patients with acute stroke by polymerase chain reaction (PCR). The blood serum of 72 patients with acute stroke and control group of 30 patients with another vascular disease was studied to reveal genom of enteroviruses. Detection of enterovirus RNA was performed by PCR using the reverse transcription. Viruses were isolated on HeLa and HEp-2 cell lines. The enterovirus genom detected by PCR was in 17 of 72 samples of the tested serum in the research group (23,6±5,0%). In the control group only one serum was PCR-positive for enteroviruses (3,3±3,2%). Cytopathogenic agents were isolated in HEp-2 and HeLa cell lines from 11 PCR-positive samples of blood serum from the experimental group of patients. No viruses were isolated from the remaining PCR-positive and PCR-negative sera. The presence of enteroviruses in the blood of patients with acute stroke suggests etiopathogenetical link between them. The introduction of PCR to detect enteroviral agents in patients with acute stroke can complement existing methods of diagnosis.

Background: Coxsackievirus A9 (CV-A9) is a pathogenic enterovirus type within the family Picornaviridae. CV-A9 infects A549 human epithelial lung carcinoma cells by attaching to the alpha V beta 6 integrin receptor through a highly conserved Arg-Gly-Asp (RGD) motif, which is located at the exposed carboxy-terminus of the capsid protein VP1 detected in all studied clinical isolates. However, genetically-modified CV-A9 that lacks the RGD motif (CV-A9-RGDdel) has been shown to be infectious in some cell lines but not in A549, suggesting that RGD-mediated integrin binding is not always essential for efficient entry of CV-A9. Methods: Two cell lines, A549 and SW480, were used in the study. SW480 was the study object for the integrin-independent entry and A549 was used as the control for integrin-dependent entry. Receptor levels were quantitated by cell sorting and quantitative PCR. Antibody blocking assay and siRNA silencing of receptor-encoding genes were used to block virus infection. Peptide phage display library was used to identify peptide binders to CV-A9. Immunofluorescence and confocal microscopy were used to visualize the virus infection in the cells. Results: We investigated the receptor use and early stages of CV-A9 internalization to SW480 human epithelial colon adenocarcinoma cells. Contrary to A549 infection, we showed that both CV-A9 and CV-A9-RGDdel internalized into SW480 cells and that function-blocking anti-alpha V integrin antibodies had no effect on the binding and entry of CV-A9. Whereas siRNA silencing of beta 6 integrin subunit had no influence on virus infection in SW480, silencing of beta 2-microglobulin (beta 2M) inhibited the virus infection in both cell lines. By using a peptide phage display screening, the virus-binding peptide identical to the N-terminal sequence of HSPA5 protein was identified and shown to block the virus infection in both A549 and SW480 cell lines. HSPA5 was also found to co-localize with CV-A9 at the SW480 cell periphery

近年来肠病毒71亚型（EV71）的大规模流行已成为全世界特别是亚太地区的一个严重的公共卫生问题。EV71感染可以引起腹泻，皮疹，手足口病等等一些自愈性疾病。然而在部分儿童患者中，EV71可能导致严重的神经性疾病。目前，关于EV71感染后宿主细胞的反应机制的报导比较少。在本次研究中，我们运用蛋白组学方法对EV71感染后的人横纹肌瘤细胞的蛋白表达情况进行了分析，最终发现了42个差异表达的蛋白（>2倍的变化，P <0.05），其中21个下调， 21个上调。进一步分析表明，这些蛋白主要参与了细胞内代谢，生物学调控，细胞构建，信息传递和细胞死亡的调控。 接下来我们选择了其中一个变化比较大的蛋白：HSP27，对其功能进行了深入分析。我们的研究结果显示：EV71感染的早期阶段，HSP27在转录和翻译水平上都有明显上调。降低HSP27表达可以减少EV71的复制，过表达HSP27则可以提高病毒复制。通过使用特异的磷酸化蛋白抗体，我们发现HSP27第15位以及78位的丝氨酸有明显的磷酸化修饰，而82位的丝氨酸则没有发生改变。使用p38激酶抑制剂预先处理细胞可以降低HSP27的磷酸化修饰，从而抑制EV71的复制。进一步分析表明，HSP27可以帮助EV71蛋白酶2A对真核翻译起始因子eIF4G的剪切，从而加强病毒自身蛋白的翻译，最终促进了病毒的感染。这项研究结果阐明了宿主细胞EV71的反应机制，有利于我们对病毒致病机制的研究，并为EV71的抗病毒研究提供了一个新的药物靶标。, The outbreaks of enterovirus 71 (EV71) infections have become a major public health issue worldwide, especially in the Asia-Pacific region. EV71 infection can be asymptomatic or cause diarrhea, rashes, and hand, foot, and mouth disease (HFMD). However, EV71 can also cause severe neurological disease even death. To date, little is known about the molecular mechanisms of the host response to EV71 infection. In this study, the expression patterns of host genes in EV71 infected human rhabdomyosarcoma cells were analyzed by using two-dimensional proteomics assays. In total, 42 protein spots were found to be differentially expressed (>2 fold changes, p<0.05) in three pairs of gels, of which 21 proteins were found to be down-regulated while 21 were up-regulated. Data analysis suggested that proteins associated with metabolic process, biological regulation, cellular component organization, cell communication and death were most modified. HSP27, one of the most altered proteins during EV71 infection, was selected to determine its fundamental roles upon EV71 infection. We show that HSP27 is rapidly up-regulated both at the transcriptional and the translational levels at the early stage of EV71 infection. Depleting cellular HSP27 expression reduced EV71 replication, while over-expression of HSP27 greatly enhanced viral infection. By using the phosphorylated specific antibodies, serine residues 15, 78, but not the 82 were found to be phosphorylated during EV71 infection. The phosphorylation depended on the activation of the mitogen-activated protein kinase p38 signaling pathway. After treating with p38 kinase inhibitors, EV71 replication was coordinately decreased. Further analysis showed that HSP27 affected the protease 2A mediated eIF4G cleavage and assisted the IRES driven translation, thus facilitated the EV71 replication. The findings in this work not only provided a global view of the host responses to EV71 infection, but demonstrated HSP27 to be a valid target for anti-E, Detailed summary in vernacular field only., Yi, Lina., "September 2012.", Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 94-103)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., s also in Chinese., p.i, 摘要 --- p.iii, Acknowledgement --- p.iv, Publications --- p.v, Table of Contents --- p.vii, List of Tables and Figures --- p.x, List of Abbreviation --- p.xii, Chapter Chapter 1 --- Introduction --- p.1, Chapter 1.1 --- Enterovirus 71 --- p.2, Chapter 1.1.1 --- Clinical features --- p.2, Chapter 1.1.2 --- Molecular epidemiology of EV71 --- p.5, Chapter 1.1.3 --- The virology of EV71 --- p.8, Chapter 1.1.4 --- Pathogenesis --- p.18, Chapter 1.1.5 --- Treatment of EV71 infection --- p.20, Chapter 1.2 --- The heat shock protein 27 --- p.23, Chapter 1.2.1 --- Properties of HSP27 --- p.23, Chapter 1.2.2 --- Functions of Hsp27 --- p.26, Chapter 1.2.5 --- Phosphorylation of Hsp27 --- p.28, Chapter 1.2.6 --- Hsp27 and Viral infection --- p.31, Chapter 1.3 --- Thesis hypothesis and objective --- p.33, Chapter Chapter 2 --- Materials and Methods --- p.34, Chapter 2.1 --- Cells and Virus propagation --- p.35, Chapter 2.2 --- Viral infection --- p.35, Chapter 2.3 --- 2-DE and image analysis --- p.36, Chapter 2.4 --- MALDI-TOF-MS --- p.37, Chapter 2.5 --- Database analysis --- p.38, Chapter 2.6 --- Bioinformatic analysis --- p.38, Chapter 2.7 --- Plasmids --- p.39, Chapter 2.8 --- siRNA synthesis --- p.41, Chapter 2.9 --- Transfection and cell treatment --- p.41, Chapter 2.10 --- RNA extraction and cDNA synthesis --- p.41, Chapter 2.11 --- Real-Time Quantitative PCR --- p.42, Chapter 2.12 --- Western Blotting analysis --- p.44, Chapter 2.13 --- Luciferase assays --- p.44, Chapter 2.14 --- Statistical Analysis --- p.45, Chapter Chapter 3 --- Proteomic analysis of cellular protein alterations in response to EV71 infection --- p.46, Chapter 3.1 --- Introduction --- p.47, Chapter 3.2 --- Results --- p.48, Chapter 3.2.1 --- EV71 infection of the RD cells --- p.48, Chapter 3.2.2 --- 2-DE profiling of EV71 infected and non-infected RD cells --- p.49, Chapter 3.2.3 --- Identification of differentially expressed proteins --- p.50, Chapter 3.2.4 --- Functional classification --- p.52, Chapter 3.2.5 --- GO enrichment analysis --- p.54, Chapter 3.2.6 --- Protein validation by Western blot --- p.56, Chapter 3.3 --- Discussion --- p.57, Chapter Chapter 4 --- HSP27 effects on EV71 infection --- p.62, Chapter 4.1 --- Introduction --- p.63, Chapter 4.2 --- Results --- p.64, Chapter 4.2.1 --- Increased Hsp27 expression in EV71 infected cells --- p.64, Chapter 4.2.2 --- Suppression of Hsp27 inhibits EV71 replication --- p.65, Chapter 4.2.3 --- Over-expression of Hsp27 increases EV71 replication --- p.66, Chapter 4.2.4 --- Hsp27 is rapidly phosphorylated during EV71 infection --- p.67, Chapter 4.2.5 --- Pathways involved in Hsp27 phosphorylation --- p.68, Chapter 4.2.6 --- Role of Hsp27 phosphorylation during EV71 infection --- p.68, Chapter 4.3 --- Discussion --- p.70, Chapter Chapter 5 --- HSP27 facilitate EV71 IRES driven translation --- p.75, Chapter 5.1 --- Introduction --- p.76, Chapter 5.2 --- Results --- p.79, Chapter 5.2.1 --- Hsp27 increase viral IRES activity --- p.79, Chapter 5.2.2 --- Hsp27 affects EV71 2A mediated eIF4G cleavage --- p.80, Chapter 5.3 --- Discussion --- p.82, Chapter Chapter 6 --- Summary and Perspectives --- p.87, Chapter 6.1 --- Summary --- p.88, Chapter 6.2 --- Perspectives --- p.89, Reference --- p.93, http://library.cuhk.edu.hk/record=b5549846, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Viruses are the most common causes of meningitis, a condition characterized by inflammation of the protective membranes that surround the brain and spinal cord. During the 10-year surveillance period, there were 3,205 confirmed cases, 724 probable cases, and 2,495 suspected cases of viral meningitis among active and reserve component members. In all three categories of cases, the most common diagnoses were meningitis due to enteroviruses; however a majority of these were unspecified enteroviruses. Nearly two-thirds (64.2%) of all cases due to enteroviral infection were hospitalized; on average, cases were hospitalized for 3.2 days. Numbers of cases peaked in late summer/early fall; and higher than average numbers of cases in 2003 reflected several outbreaks that occurred in civilian populations that year. Six states (Texas, California, Virginia, North Carolina, Florida, Georgia) reported the most cases in 2003 and overall during the period. Prevention of viral meningitis relies upon the interruption of viral transmission, e.g., thorough hand washing and disinfection of contaminated surfaces., Pub. in Medical Surveillance Monthly report, v19 n8, p1-23, Aug 2012.

Viruses are the most common causes of meningitis, a condition characterized by inflammation of the protective membranes that surround the brain and spinal cord. During the 10-year surveillance period, there were 3,205 confirmed cases, 724 probable cases, and 2,495 suspected cases of viral meningitis among active and reserve component members. In all three categories of cases, the most common diagnoses were meningitis due to enteroviruses; however a majority of these were unspecified enteroviruses. Nearly two-thirds (64.2%) of all cases due to enteroviral infection were hospitalized; on average, cases were hospitalized for 3.2 days. Numbers of cases peaked in late summer/early fall; and higher than average numbers of cases in 2003 reflected several outbreaks that occurred in civilian populations that year. Six states (Texas, California, Virginia, North Carolina, Florida, Georgia) reported the most cases in 2003 and overall during the period. Prevention of viral meningitis relies upon the interruption of viral transmission, e.g., thorough hand washing and disinfection of contaminated surfaces., Pub. in Medical Surveillance Monthly Report, v19 n8, p2-23, Aug 2012.

Lu, Jing., Thesis (Ph.D.)--Chinese University of Hong Kong, 2011., Includes bibliographical references (leaves 119-138)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b6075409, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Aims: To analyze viral etiologic agents associated with influenza-like illness (ILI) in participants reporting to one outpatient health center, one pediatric hospital, and three general hospitals in El Salvador, Honduras, and Nicaragua., Published in Influenza and Other Respiratory Viruses, 2010.

Enterovirus 71 (EV71) is a type of human virus belonging to the Enterovirus genus within the Picornaviridae family. The virus mainly causes hand, foot and mouth disease in children which sometimes lead to severe neurological complications. Outbreaks of EV71 infections are serious health threats since effective antiviral drugs or vaccines are not currently available. Therefore, development of an effective vaccine is ideal for the prevention and control of EV71 disease outbreak. The use of recombinant EV71 viral protein offers an alternative to the more risky method of using whole live attenuated or inactivated virus. Our previous study using truncated VP1 protein (VP11-100) of EV71 virus fused to a carrier protein showed strong immune response in adult rabbits. The study however, did not address the issue of its effectiveness in young animals. This factor is important since EV71 mostly infected children younger than 5 year-old. In the present study, we investigated the protectiveefficacy of NPt-VP11-100 protein against EV71 infection in a recently-developed newborn mouse model system. Prior to investigation in the newborn mouse model, we evaluated the type of immune responses developed by adult mice against NPt-VP11-100 protein. In adult mice, the protein induced high levels of anti-VP1 IgG production. Purified VP1 antigen stimulated activation, proliferation and differentiation of splenocytes harvested from the immunized mice. They also produced high levels of IFN-γ. Following determination of immune responses towards NPt-VP11-100 protein in adult mice, we performed immunization and virus challenge study in newborn mice model. Since the mice was only susceptible to EV71 infection before they are 14 day-old, only two doses of immunization were carried out. Even though the IgG produced lacked neutralization properties, immunized newborn mice were still partially protected from EV71 viral challenge. They showed high (47.4%) survival rate as compared to the control grou

Acute encephalitis syndrome (AES) is a disease condition characterized be presence of fever, and altered consciousness with or without presence of seizures or a neurological deficit. This definition is broad, and AES may be caused by a wide variety of neurotropic viruses, bacteria, protozoa, fungi or may even be non-infectious in etiology. Most AES is however considered to be due to a viral encephalitis, a condition which is common in many developing countries. The epidemiologic features of AES, especially in adults are still not well defined, and this study is first of its kind from India. This dissertation consists of three original studies and two systematic literature reviews. We first performed a retrospective chart review of all patients with fever presenting to a single hospital in central India, to determine proportions of patients with AES, and to understand diagnostic and management practices in this condition. We found that AES accounts for about 20% of all acute fevers, has a high mortality, remains frequently undiagnosed, and is mostly empirically treated as malaria. In the subsequent year we designed a descriptive study to understand the temporal and spatial profile, and survival characterictics. We also conducted a case-control study to understand risk factors for AES. We found that most AES occurs in hot and humid months of the year, and proximity to a river or stream may be associated with development of the disease. About half of all patients with AES died within 30 days of disease, and low Glasgow coma score, and need for assisted ventilation significantly predicted hazard for mortality. In our extensive laboratory testing of cerebrospinal fluid and serum samples obtained from 152 AES cases suspected of having viral encephalitis, we found 31 (17%) patients who had a confirmed viral etiology. Enteroviruses were the commonest etiology (9.2% of all AES cases) followed by flaviviruses (4.3% of all AES cases). Based on serology 16 (8.8%) patients had p

Microorganisms that are found in domestic wastewater and that can cause illness in humans include bacteria viruses protozoan cysts and helminth ova. This literature review attempts to determine whether organisms contained in a frozen sewage bulb in the Antarctic ice would survive for decades. This review briefly examines the structural differences between these organisms; examines the susceptibility of these organisms to chilling freezing thawing and frozen storage and the effect these processes have on the structural components of the organisms; and compares findings from field studies including some archeo-logical studies on the ability of these organisms to withstand natural cold environments., The original document contains color images.

The straw was designed to provide easy to carry, portable water purification to soldiers in the field. The purification process takes place in a single pass while drinking water through the straw. The purification system incorporates a multistage process consisting of a iodinated (I-5) resin, a one-micron filter and an absorption system used for pH corrections and removal of iodine residual that could affect taste. The straws were evaluated for the removal of Klebsiella terragena bacteria in general non-challenging and challenging test waters at pH 5, 7 and 9 and at 5 degrees centigrade and 25 degrees centigrade, according to EPA guidelines.

RIVM rapport:In dit rapport worden alle meetgegevens van enterovirussen in oppervlaktewater, welke dient als grondstof voor de drinkwaterbereiding, samengevat voor de periode 1978 þ 1996. De meeste metingen hebben plaatsgehad in het stroomgebied van de grote rivieren in midden Nederland , gedurende de winterperiode. Gedurende deze periode bereiken de concentraties hun maximale waarden. De enterovirusconcentratie was in de grote rivieren gemiddeld 0,4 tot 3 per liter met een maximum van 13 per liter. In de overige grote oppervlaktewateren (IJsselmeer, Haringvliet, Drentsche Aa) was de concentratie gemiddeld 0,014 tot 0,022 per liter en maximaal 0,45 per liter. Uitgaande van een maximaal toelaatbare concentratie van 1,83 x 10-7 virussen per liter in drinkwater (infectierisico van 10-4 per persoon per jaar) kan een indicatie voor de benodigde verwijdering voor de bereiding van drinkwater worden afgeleid. Deze bedraagt voor oppervlaktewater uit het stroomgebied van de grote rivieren, gemiddeld 6-7 logeenheden en maximaal 8 logeenheden. Voor de bedrijven die gebruik maken van overig oppervlaktewater als bron is de benodigde verwijdering gemiddeld 5 en maximaal 6 logeenheden., This report summarises all measured enterovirus concentrations in surface waters, which are used as source for drinking water production, for the period 1978-1996. Most of these measurements were performed in the large rivers during the winter period, when concentrations reach maximum levels. In the large rivers enterovirus concentrations were on average 0.4-3.0 per litre, reaching a maximum of 13 per litre. In other large surface waters (IJsselmeer, Haringvliet, Drentsche Aa) concentrations were on average 0.014-0.022 per litre, reaching a maximum of 0.45 per litre. An indication of the treatment required to produce safe drinking water from surface water can be derived assuming a maximum allowable concentration of 1.83 x 10-7 viruses per litre in drinking water (risk of infection of 10-4 per person per year). The required treatment was on average 6-7 log units, with a maximum of 8 log units for surface water from the large rivers. For other surface waters the required treatment amounted to 5 log units on average and 6 at maximum.

The feasibility of a novel low-cost sequence-specific nucleic acid detector of great speed and dynamic range has been examined and each system component has been tested to confidence. This detector records binding events at the surface of optical wave-guides, through coupling to the evanescent wave. The detector is made possible through innovative phase measurement techniques practiced by Visidyne. The laboratory design exceeds current detection noise threshholds and holds great promise Its components have been characterized and proven to show unparalleled sensitivity; in addition, an innovative method for improving sensitivity a thousand times is presented. The novel detection and bandpass filtering techniques exceed the state of art in evanescent-wave biosensing technology. Development of this system requires technical mastery in many disciplines; therefore, diverse concepts and data are discussed. The results suggest a likely strategy by which the system components, biochemical and electronic can be integrated to form a revolutionary new technique for chemical sensing.

In order to detect enterovirus and rhinovirus sequences as separately for the virus genera by single test of RT-PCR (reverse transcription-polymerase chain reaction), two sets of the primers were prepared covering the 5'-NTR (nontranslated region) to the VP4 in the virus genome for amplification. The sequences selected for detection included a deletion of ca. 120 b (bases) stretch in the rhinovirus genome so as to get the products separate in the electrophoretic mobilities for each virus groups. The first set of the primers consisted of P3 (5'-GGCCCCTGAATGCGGCTAAT-3') and P5 (3'-GTTCTGGGATCATTTAAGTG-5') and the second was of P4 (5'-ACTTTGGGTGTCCGTGTTTC-3') and P5；the P3 and P4 (for a closer site to the P5 compared to the P3 site) were complementary to the sequences in the 5'-NTR of 95～100% homology and P5 being in the VP4 region complementary to the sequence of 60～90% homology through the two virus groups, each. By considering the relatively low homology in the counterpart site, the P5 primer was employed for initiation of the reverse transcription at a low temperature of 37℃ for promoting the primer annealing to the template RNA. When applied to the prototype strains, the P3-P5 primers gave a single band equivalent to 460 b DNA for the enteroviruses (43/44 serotypes) and that of 340 b DNA for the rhinoviruses (8/8 serotypes), thus enabling to easily discriminate the two virus groups on the separate mobilities of the products. Similarly, the products by the P4-P5 primers were of 340 b and 220 b DNAs for enteroviruses (43/44 serotypes) and rhinoviruses (8/8 serotypes), respectively, again being of easy discrimination of the virus groups. Of these experiments, authenticity of the products was confirmed by base sequence analysis with selected product samples from the experimental groups. The primers prepared were also compared for the efficiencies of virus detection in the clinical specimens with the tissue culture method for virus isolation. Positive ratios resulted w

RIVM rapport:In een kwantitatieve beschrijving van de emissie en verspreiding van Cryptosporidium, Giardia en enterovirussen in de Nederlandse oppervlaktewateren met behulp van emissie- en verspreidingsmodellen zijn concentraties van genoemde pathogenen geschat. De emissies werden met PROMISE gemodelleerd, uitgaande van gemiddelde concentraties in ongezuiverd afvalwater en gemiddelde verwijdering bij afvalwaterzuivering. De emissie van Cryptosporidium was grotendeels afkomstig van gezuiverde lozingen (84%), terwijl emissie van Giardia en enterovirussen grotendeels afkomstig was van ongezuiverde lozingen (respectievelijk 82% en 69%). Verspreiding in het oppervlaktewater werd berekend met WATNAT met voor Cryptosporidium een constante inactivatie-snelheid en voor Giardia en enterovirussen een temperatuurafhankelijke inactivatie-snelheid. Op verschillende locaties in de grote rivieren was er een goede overeenstemming tussen gemeten en berekende concentraties van de protozoa. Voor enterovirussen was dit op vrijwel alle locaties het geval. Volgens de schattingen had echter de buitenlandse aanvoer van pathogene micro-organismen een overheersende invloed op de concentraties in het oppervlaktewater van de grote rivieren. De concentraties protozoa vanuit Duitsland waren in orde van grootte gelijk aan die vanuit Belgie, hetgeen een indicatie is voor de aanwezigheid van andere emissiebronnen dan huishoudelijk afvalwater, zoals uit- en afspoeling van mest van landbouwhuisdieren. De benodigde verwijderingen van Cryptosporidium, Giardia en enterovirussen voor de bereiding van drinkwater werden geschat op 5 tot 8 logeenheden. Deze modellering vormt de basis voor berekening van de blootstelling aan deze micro-organismen ten gevolge van het gebruik van oppervlaktewater voor recreatie en drinkwater. In combinatie met dosis-response relaties kunnen gezondheidsrisico's worden geschat. Deze modellen kunnen ook worden toegepast om de effecten van maatregelen te kwantificeren en om volgens bepaalde sce, A quantitative description is presented on the emissions and distribution of the pathogens mentioned through domestic waste water. The emissions were modelled with PROMISE based on average concentrations in untreated wastewater and the average removal by sewage treatment. Emission of Cryptosporidium originated largely from discharges of treated wastewater (84%), whilst emission of Giardia and enteroviruses came for the larger part from discharges of untreated wastewater (respectively 82% and 69%). Distribution in surface waters was calculated with WATNAT assuming a constant inactivation rate of Cryptosporidium and a temperature dependent inactivation rate for Giardia and enteroviruses. At several locations in the large rivers actual measured and calculated concentrations of the protozoa were similar. For enteroviruses this was the case at almost all locations. It was, however, estimated that discharges from abroad were the major source for the occurrence of these pathogens in the large rivers. The concentrations of protozoa coming from Germany were of similar level as those coming from Belgium, which indicate at the existence of other sources of emission than domestic wastewater, such as run-off of animal manure from land. The required treatment of Cryptosporidium, Giardia and enteroviruses for the production of drinking water were estimated to be 5 to 8 log units. Concentrations of pathogens in surface waters on a national scale could be estimated with models, which form the basis for calculation of exposure to these micro-organisms due to water recreation and drinking water consumption. Combined with dose-response relations health risks can be estimated. These models can also be applied to quantify the effects of measures and to make prognoses on the quality of surface waters and the related health risks according to certain scenario's.

Tests were conducted inhouse to determine the microbiological treatment efficacy of the Pre-Mac Model FWP hand-held water purifiers. Test waters represented various physical/chemical challenge conditions. Microbial challenges consisted of enteric bacteria (Klebsiella terrigena), enteroviruses (Echovirus 1 and Poliovirus 1), protozoan oocysts (Crytosporidiu parvum), and cyst simulant (latex beads). Additional tests were conducted at the University of Arizona (UA) to determine the viability of Cryptosporidium parvum oocysts after exposure to the Pre-Mac iodine iodine, and to evaluate Pre-Mac water purifiers fitted with second generation 1.0 micron fine mesh filters for physical removals of cysts from product water. Studies used the U.S. Environmental Protection Agency's interim Guide Standard and Protocol for Testing Microbiological Water Purifiers for guidance. Results indicated that bacteria and viruses were readily removed by the Pre-Mac filter units. However, test results from inhouse studies and UA studies indicated that the Pre-Mac water purifiers were not adequate to physically remove or reduce infectious levels of protozoan cysts to meet the 3-log reduction criteria, Prepared in collaboration with Arizona Univ., Tucson. Dept. of Veterinary Science.

A method of adsorbing enteroviruses to bentonite clay was developed for use as a concentration technique designed to sample low levels of virus in environmental and tap waters. A divalentcation such as calcium was required to enhance the adsorption of large quantities of poliovirus onto bentonite within a 20 minute contact period. A minimum bentonite level of 50 mg/L was necessary to adsorb the virus and to still allow efficient recovery and reasonable sample - size collection onto Miliporetm AP25 prefilters. Virus elution from the clay concentrated on the AP25 prefilters was most efficient with a solution mixture containing glycine (0.1 M), EDTA (0.01 M), and 1% bovine serum at pH 11, although tryptose phosphate broth at pH 7.2 was favored for field sampling with long field storage or shipping requirements. A water pH range of 4-10 did not significantly alter the efficiency of virus recovery. Although best recovery was obtained from highly polished water, good recoveries were also found for tap and wastewater with virus recoveries of 4585%. The technique was found to be very versatile and could be easily scaled up for larger sample volumes without a loss in recovery efficiency. Enterovirus, Virus Concentration, Adsorption, Elution, Bentonite, Virus Sampling.

The objectives of this work are to further our knowledge of the pathogenesis of hepatitis A virus (HAV) infection in man, and to develop recombinant expression vectors for hepatitis A virus antigens that can be used to stimulate mucosal immunity. Two viral cDNA sequences encoding different forms of capsid proteins have been successfully cloned into several bacterial expression vector plasmids, transformed into E. coli and analyzed. Expression tests are still in progress. An in situ hybridization procedure has been developed, using probes developed in our cloning experiments. This procedure can readily detect HAV-infected cultured cells, and is now ready to be applied to fixed animal tissue sections. HAV is being grown in small scale in our laboratory, and a 'dot blot' hybridization procedure has been developed to understand the course of infection. A variety of human acute and convalescent sera have been obtained for analysis of the anti-HAV antibody specificity by immunoblot tests against virus, viral capsid proteins and infected cell extracts. These experiments should help advance our understanding of the pathogenesis of enteric virus infections and begin to elucidate new strategies for the construction of viral vaccines.

An improved method to identify human rotavirus in stool samples was developed through preparation and use of a monoclonal antibody reagent directed against the group specific antigen shared by mammalian rotaviruses. The monoclonal antibody reagent has been adapted to an enzyme-linked immunosorbent assay (EIA), making it available for use in field studies. This monoclonal EIA has considerably improved the sensitivity and specificity of rotavirus EIA detection efforts with neonatal stool samples and adult stool specimens. We are also continuing to develop monoclonal antibodies against the to-date noncultivatable Norwalk virus both by production of human-mouse heterohybridomas and by immunization of mice with extensively purified Norwalk virus. Recently, we began to develop a much needed convenient, quantitative immunoassay for the study of enteric adenoviruses (types 40 and 41)in spite of current difficulties in their recognition in vitro. We reported severe diarrhea produced by rotavirus in adults requiring hospitalization in Thailand, as well as evidence implicating rotavirus as a cause of waterborne gastroenteritis in the U.S. Six U.S. outbreaks of gastroenteritis associated with the consumption of raw and undercooked clams and oysters were shown to be produced by Norwalk virus; one Norwalk virus outbreak was associated with eating food from a salad bar.

Studies on the virucidal properties of seawater indicate that microbial agents are involved since (a) both autoclaving and filtration remove the activity and (b) the activity can be restored to the filtrate if the filter pads are immersed back in the filtrate. Studies on high temperature (62 C) inactivation indicate that all 3 enteroviruses were inactivated within 30' at pasteurization temperatures. Studies of the effects of temperature and salinity on the survival of three enteric viruses (Poliomyelitis type 1, echovirus-6, and coxsackievirus B-5) under controlled laboratory conditions and in situ indicate that temperature rather than salinity is the critical factor affecting their stability, in that the higher the temperature the more rapid was the loss of viral infectivity. In the laboratory studies all three viruses were quite stable at 4 C, with infectious virus still detectable after 46 weeks of incubation. In situ studies on virus survival in free-flowing estuarine or marine waters showed that, although the viruses were more labile in natural waters than in the laboratory studies, they persisted for several months, in some cases during the winter months. At all tmeperatures and salinities, coxsackievirus B-5 was the most stable, echovirus-6 was intermediate, and poliovirus 1 was the least stable of the viruses tested.

In closed living and community rooms, many viruses can long retain infectious activity in air. The more stable under these conditions are the adenoviruses, the enteroviruses and the Newcastle's disease virus, but the parainfluenza viruses and respiratory-syncytial virus are inactivated sufficiently rapidly. The relative humidity has a significant effect on the degree of virus inactivation in the air., Edited machine trans. of Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii (USSR) n4 p21-25 1973, by Michael L. Seidel.

Supersedes AD-A129 542. Presented in part at the poster session, ASCP-CAP 1982 Joint Spring Meeting Held on 13-18 Mar 82 at New Orleans, LA.

The purpose was to determine extent that individuals near spray irrigation sites are exposed to microorganisms in wastewater aerosols. Report covers Phase II - a monitoring effort from May 1976 to April 1977 of a spray irrigation site utilizing unchlorinated secondarily-treated wastewater from biofiltration treatment process. Objectives included an in-depth pathogen screen of wastewater, establishing the relationship between pathogen levels and traditional indicator organisms, monitoring microorganisms in air within 600 meters of the spray source, and development/validation of a microbiological dispersion model for predicting pathogen aerosol concentrations. Effluent was monitored for microbiological, chemical, and physical characteristics; extensive microorganism and dye aerosol samples were collected (77 aerosol runs). Enteroviruses were detected in air, but at a very low concentration. Conclusions: There is considerable underestimation of pathogen aerosol levels when using traditional indicators to predict human exposures to enteroviruses. Microbiological dispersion model may be used with minimal monitoring to estimate exposure. There is little correlation between wastewater levels of traditional indicators and pathogens. Microbiological wastewater aerosols are generated by spray irrigation, do survive aerosolization and can be transported to nearby populations.

Survival studies in filtered seawater at varying temperatures showed an increase in parvovirus viability with decreasing temperature. In further investigation of enterovirus elimination from untreated seawater, a marine bacterium, Acinetobacter calcoaceticus, was isolated and found to possess a poliovirus I, non-filterable, virucidal effect. Survival of coliforms and human enteric pathogens in Chesapeake Bay also has been examined. (Author)

Experimental data obtained from field models since 1975 have shown that virus removal in holding ponds involves two components: inactivation and sedimentation. Several factors may be cited as contributing to viral inactivation. Undoubtedly, higher temperatures can lead to a more rapid inactivation of poliovirus. Such a primary effect is seen in both laboratory controls and in the differences between winter and spring field results. A secondary influence of temperature and sunlight, however, is monitored in terms of increased biological activity. While the cumulative effects of various microbiotic communities are difficult to dissect under field conditions, general biological activity is seen as antagonistic to viral populations. Another important field observation was the appearance of poliovirus in the sediments generated during the life of the model holding ponds. Initial detection of infectious viruses in sediments during the winter run led to a more detailed sediment monitoring system for the spring tests.

A rapid method for the isolation and identification of viruses from water has been developed. This method allows for the demonstration of viruses in the water sample after only 6-9 hours of incubation, whereas, a time interval of 18-24 hours should satisfy most requirements for water distribution. One must take into account, however, the possibility of 'false negatives' or 'false positives' in the high speed determinations, probably due to impurities of the reagent used in the technique.

The identification of untypable viruses isolated in the Cairo Infantil Diarrhea Study is completed. Some were identified as recognized known enteroviruses, but most of them proved to be 'prime strains' of echovirus types 12 through 33.

It is apparent from the results of the virus studies that enteric viruses survive for relatively long periods of time in estuarine and marine salinities when the water temperature is 25C or lower. Of the three enterioviruses tested, Coxsackie B-5 virus has been the most stable one with ECHO-6 virus being intermediate and polio 1 the least stable virus. This was true both in controlled laboratory experiments and in situ and is probably indicative of the diversity in stability one would encounter if all of the 100+ human enteroviruses were studied. While the salinity of the water may have minor effects on enterovirus stability, clearly the most important factor affecting virus viability was the temperature of the water.

The studies reported have a direct relationship to medical problems of military importance with particular reference to the tropics. The medical problems which have been studied include the etiologies of diarrheal diseases in the tropics, pathogenesis of amebic lesions, etiology of infectious hepatitis, etiology of acute respiratory disease in Costa Rica, biological control of Chagas' disease vectors, reconnaissance for arbovirus activity in the Mississippi River delta region, taxonomy, distribution, and ecology of phlebotomine flies in Costa Rica, clinical importance of prothrombin time in snake venom poisoning, pathophysiology and treatment of septic shock, skin test for hookworm infection, and enteroviruses in rural families and their animals., See also report dated 30 Oct 72, AD-757 178.

Enteroviruses affect a broad segment of the population throughout the world and have been suspected to play a major role in waterborne disease for quite some time. The presence of these viruses in drinking water supplies constitutes a major health risk to the population because of their low infectious dose. The monitoring and study of these viruses in the environment have been limited by the current standard detection methodologies. Nucleic acid probe hybridization is a new and effective approach for the study and detection of these viruses in the environment. An important step in the detection of viruses in concentrated water samples by nucleic acid probes is the isolation of the viral genome from the water sample for hybridization. Previously, a series of time consuming organic extract ions was used to isolate viral RNA. This study reports the development of an alternative method for the isolation and preservation of viral RNA in environmental samples. Briefly, the sample is heated in the presence of an RNase inhibitor, and then applied to a hybridization membrane. This procedure has greatly reduced the time and difficulty of the assay while maintaining sensitivity and increasing consistency. This study reports the development and modification of a nonradioactive labeling system for the detection of viruses in water. Nonradioactive labels such as biotin offer several advantages over radioactive labels including unlimited shelf life, reduced cost and time of assay, and elimination of the radiation hazard. However, radioactive labels are generally the more sensitive method of detection. By combining direct and indirect labeling strategies, the sensitivity of this nonradioactive assay has been increased ten-fold. This assay can detect as little as 100 plaque forming units of poliovirus, only one order of magnitude less sensitive than radiolabeled probes. This assay is also ten-fold less sensitive than radiolabeled probes for the detection of enteroviruses in water sa

Many isolations of coxsackie viruses B1, B3 and B5 and echovirus 6, related if not identical to human viruses of the same types were made from nose, throat and rectal swabs of Beagle dogs. Six unidentified enteroviruses were also recovered. Except for the echovirus 6 such isolations have not been previously reported. Three groups of Beagles receiving an aerosol which provided an initial body burden of 90 90 144 35 micronCi/kg Sr, 90 MicronCi/kg Sr and 140 ;micronCi/kg Ce showed no difference in the incidence of coxsackie B viruses from that of controls. However, a significantly greater number of isolations of echovirus 6 and of an unidentified enterovirus were made from controls than from exposed dogs. Low NT antibody titres against coxsackieviruses B3 and B5 were found in some of the serum from the Beagles that were examined. No titres were observed for coxsackievirus B1, echovirus 6 or one of unidentified enterovirus which were the most frequently isolated. There was no correlation between virus isolation and serum titres.

Fecal specimens from apparently healthy dogs were inoculated into MK, DK, HeLa, and OP cells. Twenty-six cytopathic agents were isolated in MK, ii in HeLa, and 4 in OP, but none in DK cells. Neutralization tests indicated that all but one of the viruses were either strains of ECHO type 6 or were closely related to it. Twenty-one of 29 dogs were carrying the virus., ADA383784 ADA383785