Teorico 3 - Resumen De Clase De Gastroenteritis Virales 2005

Resumen de la clase teórica sobre: GASTROENTERITIS VIRALES Dictada el día 8 de agosto de 2005. Prof. Dra. Liliana Martínez Peralta Las gastroenteritis son procesos inflamatorios con o sin disfunción del intestino producidas por un germen o sus toxinas. Se caracterizan por un síndrome diarreico agudo acompañado o no de vómitos y dolor abdominal. Las gastroenteritis virales se encuentran entre las enfermedades más comunes que afectan al hombre; y su morbilidad y mortalidad son mayores en los extremos de la vida. Se calcula que son la principal causa de mortalidad infantil, calculándose entre 5 á 10 millones de muertes anuales. En países desarrollados, si bien la mortalidad es menos frecuente, las gastroenteritis requieren hospitalización, siendo la principal causa de deshidratación severa y de importante morbilidad. La gran mayoría de las gastroenteritis virales son causadas por virus pertenecientes a 4 familias: Rotavirus (Reovirus); Calivirus; Adenovirus y Astrovirus. Otros virus como Torovirus, Picornavirus, Picobirnavirus pueden ser agentes causales, con distribución menor. Todos estos virus infectan al hombre a través del tracto alimentario, que opone a estos agentes dificultades tales como la acidez gástrica, la alcalinidad del intestino, las enzimas digestivas, sales biliares y los mecanismos inespecíficos de defensa. Los virus que evolucionaron para infectar por esta ruta son desnudos, con la única excepción de los Coronavirus. El modo de transmisión de estos virus es fecal-oral, por la contaminación del agua y los alimentos, el ciclo ano-mano-boca en la 1era infancia y algunas prácticas sexuales en adultos. Las gastroenteritis virales presentan 2 patrones epidemiológicos característicos: 1) casos endémicos de diarrea en niños: ocurren a lo largo del año con mayor prevalencia en meses templados y fríos, afectando fundamentalmente a niños de 1era infancia (hasta 3-4 años) siendo importantes las condiciones sanitarias. La causa más frecuente de estos cuadros son los Rotavirus, siguiendo como causa los Astrovirus, Adenovirus entéricos y algunos Calicivirus. 2) Brotes epidémicos de diarreas virales que afectan a sujetos de todas las edades, generalmente a través de agua y comida contaminada. Son causados principalmente por virus del género Norwalk (Calicivirus). Por ser el principal responsable de las gastroenteritis virales se referirá con mayor extensión a los rotavirus. Los rotavirus son miembros de la familia Reoviridae: desnudos, icosaédricos y tienen genoma con ARN de cadena doble y segmentado (los rotavirus humanos presentan 11 segmentos). Al microscópio electrónico presentan aspecto de rueda, que les da su nombre en latín. Cada segmento del genoma codifica para una de las proteínas virales (6 estructurales y 6 no estructurales), con excepción del segmento 11 que codifica para 2. Los estudios de criomicroscopía electrónica han permitido demostrar la estructura complicada de estos virus: tienen tres capas concéntricas formando la cápside viral. La capa más externa está formada por la glicoproteína VP7 y la proteína VP4, que forma las espículas externas. La VP4 se cliva por un proceso proteolítico originando 2 proteínas VP5 y VP8 que vuelven infecciosa a la partícula viral. La capa del medio está formada por VP6; que es esencial para la expresión del genoma viral. Posee unos canales por los que saldrán los ARNm virales y sufre cambios conformacionales de acuerdo a su transcripción endógena. La capa interna está formada por 3 proteínas estructurales: VP1; VP2 y VP3. De ellas VP2 forma el andamiaje proteico que interacciona con VP6 hacia fuera y con el ARN genómico en el interior. VP1 es una ARN polimerasa y VP3 es una guanil-metil- transferasa que forman el complejo de transcripción para cada segmento genómico viral. Replicación del virus: Las partículas se vuelven infecciosas cuando se cliva VP4 por las proteasas del intestino. Su tropismo es por los enterocitos maduros del intestino delgado. La VP8 se une a residuos de ácido siálico y la VP5 a moléculas de integrinas, como αvβ3. La penetración sería a través de la traslocación directa, o bien por un mecanismo alternativo de endocitosis que involucra a la Dinamina. Una vez en el citoplasma se produce el denudamiento de la cubierta proteica externa, liberándose partículas con dos capas que son las unidades funcionales que producen los ARNm virales. La transcripción de mensajeros y la síntesis de nuevas copias de genoma viral se lleva a cabo íntegramente dentro de la partícula viral. Los ARNm pasan a través de los poros de la capa media para la síntesis de proteínas en el citoplasma celular. La síntesis de proteínas virales a partir de los mensajeros virales es facilitada por la proteína viral NSP3 que se fija a estos mensajeros en el extremo 5’. Esta proteína les da una ventaja competitiva a los mensajeros virales y evita que las nucleasas celulares los degraden. El proceso de empaquetamiento y replicación del genoma viral se lleva a cabo en inclusiones citoplasmáticas llamadas viroplasmas. Para el ensamblado final de la cápside trilaminar se lleva a cabo en el retículo endoplásmico. El egreso de la progenie viral se lleva a cabo por la lisis de la célula infectada. Mecanismo de producción de diarrea: En síntesis, los rotavirus pueden provocar diarrea posiblemente por 3 mecanismos: 1) Malabsorción secundaria a la destrucción de entericitos con disminución del área de absorción y reemplazo por células inmaduras funcionalmente incompetentes. 2) Secreción de Cl- (acompañado de Na+ y H2O) por efecto de una enterotoxina viral: NSP4. 3) Activación del sistema nervioso entérico con el aumento del péptido intestinal vasoactivo y su acción secretagoga. Después de una primoinfección con rotavirus los niños presentan una protección limitada frente a reinfecciones, que son frecuentes pero más moderadas.

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Se clasifican en grupos (A-B-C-D-E-F-G-) y subgrupos ( [1] á [4]) según la reacción serológica. Así, el grupo A, que es el más frecuente agente causal de diarreas en el hombre, se clasifica en 4 subgrupos, que a su vez se subclasifican en tipos virales según la variante de proteína VP7 (G=14) y de la proteína VP4 (P=20). Así un virus se denomina como rotavirus A [1] G1P1. Los electroferotipos sirven para estudios epidemiológicos dada la alta variabilidad de estos virus. En los estudios epidemiológicos de nuestro país se ha encontrado que predominan los tipos G1; G2 y G4. La incidencia es mayor en las provincias del noroeste. La vacuna ensayada en EEUU a virus vivo y atenuado fue suspendida por asociarse con un importante número de casos con invaginación intestinal. Se están desarrollando y probando nuevas vacunas. Astrovirus: Son virus extremadamente simples, desnudos y con cápsides icosaédricas formadas por una sola proteína. Su genoma es un segmento de ARN +. Son la 2da causa en frecuencia de gastroenteritis virales en lactantes pero ocasionalmente pueden causar brotes en sujetos mayores. Adenovirus entéricos: Son fundamente patógenos respiratorios, tienen una cápside icosaédrica, son desnudos y poseen un genoma ADN de doble cadena lineal. Sólo dos serotipos (40 y 41) pueden causar gastroenteritis y a su vez son incapaces de producir enfermedad respiratoria. Esta infección puede ser más prolongada que las de los otros virus. Calicivirus: Son desnudos, icosaédricos y genoma ARN+. Estos virus son los principales responsables de los brotes epidémicos de gastroenteritis virales que afectan a niños y adultos. Con un período de incubación de aproximadamente 12 horas, la enfermedad se limita a 2-3 días, y predominan las náuseas y vómitos sobre la diarrea. Diagnóstico general de gastroenteritis: Para el diagnóstico del agente etiológico existe la posibilidad de detectar antígenos virales en materia fecal por equipos comerciales de ELISA o aglutinación de partículas. Métodos más sofisticados como M/E y PCR se reservan a centros de referencia y control epidemiológico de la población. Bibliografía sugerida Bereciartu A, Bok K, Gomez J. Identification of viral agents causing gastroenteritis among children in Buenos Aires, Argentina. J Clin Virol. 2002 Aug;25(2):197-203. http://www.novartisfound.org.uk/catalog/238abs.htm Gomez JA, Sordo ME, Gentile A. Epidemiologic patterns of diarrheal disease in Argentina: estimation of rotavirus disease burden. Pediatr Infect Dis J. 2002 Sep;21(9):843-50. Lundgren O, Svensson L. Pathogenesis of rotavirus diarrhea. Microbes Infect. 2001 Nov;3(13):1145-56. Review. Morris A & Estes MK. Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interactions. VIII Pathological Consequences of rotaviruse infection and its enterotoxin. Am. J Physiol Gastrointest Liver Physiol. 2001 281:G303-310.

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Abstracts

Gastroenteritis viruses: an overview Roger I. Glass, Joseph Bresee, Baoming Jiang, Jon Gentsch, Tamie Ando, Rebecca Fankhauser, Jacqueline Noel, Umesh Parashar, Blair Rosen and Stephan S. Monroe Viral Gastroenteritis Section, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA Acute gastroenteritis is among the most common illnesses of humankind, and its associated morbidity and mortality are greatest among those at the extremes of age, children and the elderly. In developing countries, gastroenteritis is a common cause of death in children <5 years that can be linked to a wide variety of pathogens. In developed countries, while deaths from diarrhoea are less common, much illness leads to hospitalization or doctor visits. Much of the gastroenteritis in children is caused by viruses belonging to four distinct families-rotaviruses, caliciviruses, astroviruses and adenoviruses. Other viruses, such as the toroviruses, picobirnaviruses, picornavirus (the Aichi virus), and enterovirus 22 and 23, may play a role as well. Viral gastroenteritis occurs with two epidemiologic patterns, diarrhoea that is endemic in children and outbreaks that affect people of all ages. Viral diarrhoea in children is caused by group A rotavirus, enteric adenovirus, astrovirus and the caliciviruses; the illness affects all children worldwide in the first few years of life regardless of their level of hygiene, quality of water, food or sanitation, or type of behaviour. For all but perhaps the calicivir uses, these infections provide immunity from severe disease upon reinfection. Epidemic viral diarrhoea is caused primarily by the Norwalk-like genus of the caliciviruses. These viruses affect people of all ages, are often transmitted by faecally contaminated food or water, and are therefore subject to control by public health measures. The tremendous antigenic diversity of caliciviruses and shortlived immunity to infection permits repeated episodes throughout life. In the past decade, the molecular characterization of many of these gastroenteritis viruses has led to advances both in our understanding of the pathogens themselves and in development of a new generation of diagnostics. Application of these more sensitive methods to detect and characterize individual agents is just beginning, but has already opened up new avenues to reassess their disease burden, examine their molecular epidemiology, and consider new directions for their prevention and control through vaccination, improvements in food and water quality and sanitary practices.

Structural studies of gastroenteritis viruses B. V. Venkataram Prasad*, S. Crawford†, J. A. Lawton*1, J. Pesavento*, M. Hardy†2, and M. K. Estes† *Verna and Maars McLean Department of Biochemistry and Molecular Biology, and †Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA There are many recent advances in our understanding of the structure-function relationships in rotavirus, a major pathogen of infantile gastroenteritis, and Norwalk virus, a causative agent of epidemic gastroenteritis in humans. Rotavirus is a large (1000 Å) and complex icosahedral assembly formed by three concentric capsid layers that enclose the viral genome of 11 dsRNA segments. Because of the medical relevance, intriguing structural complexity, and several unique strategies in the morphogenesis and replication, this virus has been a subject of extensive biochemical, genetic and structural studies. Using a combination of electron cryomicroscopy, computer image processing together with atomic resolution X-ray structural information, we have been able to provide not only a better description of the rotavirus architecture, but also a better understanding of the structural basis of various biological functions such as trypsin-enhanced infectivity, virus assembly and the dynamic process of endogenous transcription. In contrast to rotavirus, Norwalk virus has a simple architecture with an icosahedral capsid made of 180 copies of a single protein. We have determined the structure of the Norwalk virus capsid to a resolution of 3.4 Å using X-ray crystallographic techniques. These studies have provided valuable information on domain organization in the capsid protein, and residues that may be critical for dimerization, assembly, strain-specificity and antigenicity.

Early events of rotavirus infection: the search for the receptor(s) Carlos F. Arias, Carlos A. Guerrero1, Ernesto Méndez, Selene Zárate, Pavel Isa, Rafaela Espinosa, Pedro Romero and Susana López

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Departamento de Genética y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62250, Mexico The entry of rotaviruses into epithelial cells seems to be a multistep process. Infection competition experiments have suggested that at least three different interactions between the virus and cell surface molecules take place during the early events of infection, and glycolipids as well as glycoproteins have been suggested to be primary attachment receptors for rotaviruses. The infectivity of some rotavirus strains depend on the presence of sialic acid on the cell surface, however, it has been shown that this interaction is not essential, and it has been suggested that there exists a neuraminidase-resistant cell surface molecule with which most rotaviruses interact. The comparative characterization of the sialic acid-dependent rotavirus strain RRV (G1P1A[8]), its neuraminidase-resistant variant nar3, and the human rotavirus strain Wa (G3P5[3]) has allowed us to show that a2b1 integrin is used by nar3 as its primary cell attachment site, and by RRV in a second interaction, subsequent to its initial contact with a sialic acid-containing cell receptor. We have also shown that integrin aVb3 is used by all three rotavirus strains as a co-receptor, subsequent to their initial attachment to the cell. We propose that the functional rotavirus receptor is a complex of several cell molecules most likely immersed in glycosphingolipid-enriched plasma membrane microdomains.

Rotavirus RNA replication and gene expression John T. Patton Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, 7 Center Drive, MSC 0720, Room 117, Bethesda, MD 20892, USA Rotavirus mRNAs are capped but non-polyadenylated and serve as templates for both the synthesis of viral proteins and the segmented dsRNA genome. Viral proteins involved in RNA replication include the RNA polymerase (VP1), the core scaffold protein (VP2) and the non-structural RNA-binding proteins (NSP2 and NSP5). VP2 enhances dsRNA synthesis in vitro, possibly by forming platform structures on which VP1 functions. NSP2 octamers have NTPase and helix-destabilizing activity, and in conjunction with the phosphoprotein NSP5, are proposed to facilitate RNA packaging. The structure of the mRNA template contributes importantly to RNA replication. In particular, base-pairing between the 5' and 3'-ends of viral mRNA generates panhandle structures which promote minusstrand synthesis. For the group A rotaviruses, the 3'-consensus sequence, 5'-UGUGACC-3', which extends as a 3'-tail from the panhandles, also contributes to efficient minus-strand synthesis. Besides containing cis-acting replication signals, the 3'-end of viral mRNAs contains information that stimulates gene expression in infected cells. Specifically, the last four nucleotides of the 3'-consensus sequence, 5'-GACC-3', operate as a viral-specific translation enhancer (3'TE) via a process thought to involve recognition of the element by NSP3. The NSP3-3'TE complex may mimic the function of complexes formed by eukaryotic poly(A)-tails and poly(A)binding protein, thereby promoting more efficient translation of viral mRNAs.

Pathogenesis of rotavirus gastroenteritis Mary K. Estes, Gagandeep Kang, Carl Q.-Y. Zeng, Sue E. Crawford and Max Ciarlet Division of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA The outcome of intestinal infection with rotaviruses is more complex than initially appreciated, and it is affected by a complex interplay of host and viral factors. Rotaviruses infect intestinal enterocytes, and the early events in infection are mediated by virus-epithelial cell interactions. Diarrhoea may be caused by several mechanisms including (i) malabsorption that occurs secondary to the destruction of enterocytes, (ii) villus ischaemia and activation of the enteric nervous system that may be evoked by release of a vasoactive agent from infected epithelial cells in the absence of significant pathologic lesions or enterocyte damage, and (iii) intestinal secretion stimulated by the intracellular or extracellular action of the rotavirus non-structural protein, NSP4, a novel enterotoxin and secretory agonist with pleiotropic properties. New studies of rotavirus infection of polarized intestinal epithelial cells show that rotaviruses infect cells differently depending on whether or not they require sialic acid for initial binding, and infection alters epithelial cell functions. NSP4 also affects epithelial cell function and interactions. NSP4 (i) induces an age- and dose-dependent diarrhoeal response in young rodents that is similar to virus-induced disease, (ii) stimulates a Ca2+-dependent cell permeability where the secretory response is age-dependent, and (iii) alters epithelial cell integrity. Antibody to NSP4 protects mouse pups from diarrhoea induced by homotypic and heterotypic viruses. These data support a new mechanism of rotavirus-induced diarrhoea whereby a viral enterotoxin triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. This new information about how a gastrointestinal virus causes disease demonstrates common pathogenic mechanisms for viral and bacterial pathogens not previously appreciated. These results also suggest new approaches to prevent or treat rotavirus-induced diarrhoea.

Correlates of protection against rotavirus infection and disease

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Paul A. Offit The Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., The University of Pennsylvania School of Medicine Philadelphia, PA 19104, and The Wistar Institute of Anatomy and Biology, 36th and Spruce Sts, Philadelphia, PA 19103, USA Repeated infections with the 'mucosal' pathogen rotavirus are common in children. Subsequent rotavirus infections usually cause milder symptoms than first-time infections. Therefore, although natural rotavirus infection attenuates the severity of subsequent infections, it does not prevent reinfection or mild disease. On the other hand, natural infection with 'systemic' viruses such as measles, mumps, rubella, or varicella often confers life-long protection against mild disease associated with reinfection. The degree to which differences in the pathogenesis of systemic and mucosal pathogens determines differences in the capacity of natural infection to induce life-long protective immunity will be discussed. This paradigm will be used to explore the immunological effector functions associated with protection against rotavirus challenge.

Rotavirus epidemiology and surveillance Ulrich Desselberger, Miren Iturriza-Gómara and Jim J. Gray Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge, CB2 2QW, UK There is extensive antigenic and genomic diversity among co-circulating human rotaviruses. They are differentiated into groups, subgroups and types. There are at least 7 groups (A-G) and 4 subgroups within group A. To distinguish types within group A, a dual classification system has been established with the glycoprotein VP7 defining G types, and the protease-sensitive protein VP4 defining P types. At least 14 G types and more than 20 P types have been distinguished, of which at least 10 G types and at least 11 P types have been found in humans. Using the typing system, we have investigated the complex molecular epidemiology of rotaviruses. Rotaviruses of different G and P types co-circulate. The main types found are G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8]; their relative incidence rates change over time in any one location and are different at the same time between different locations. Viruses with G/P constellations such as G1P1B[4] and G2P1A[8] are often natural reassortants of the co-circulating main virus types emerging after double infection of hosts. Viruses carrying G and or P types not represented in the four most common types, e.g. G8P[8], G1P[6] or G9P[6], could be introduced into the population by reassortment with animal viruses, or directly from animals or exotic human sources. Naturally circulating rotaviruses constantly undergo point mutations which can be used to classify lineages and sublineages within types. The full significance of human infections with group B and C rotaviruses remains to be established. Surveillance of rotavirus types in different parts of the world is essential to monitor the emergence of new types or of new G/P constellations which may predominate over time. The efficacy and effectiveness of any future rotavirus vaccine may differ depending on the predominant natural strain types. Detailed epidemiological and molecular surveillance data should be utilized to study the transmission dynamics of rotaviruses.

A rotavirus vaccine for prevention of severe diarrhoea of infants and young children: development, utilization and withdrawl Albert Z. Kapikian Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20852, USA The importance of rotaviruses (RVs) as the single most important cause of severe diarrhoea of infants and young children is well recognized. At NIH, we developed a quadrivalent (tetravalent [TV]) vaccine to protect against the four epidemiologically important RV serotypes. It is comprised of live attenuated rhesus RV (RRV), a VP7 serotype G3 strain (the 'Jennerian' approach), and three reassortant RVs, each containing 10 RRV genes and one human RV gene that codes for the major outer protein, VP7, that determines serotype G1, G2 or G4 specificity (the 'modified Jennerian' approach). The vaccine was safe and effective against severe diarrhoea in a major pre-licensure collaborative effort of phase III trials. In June 1998, the Advisory Committee on Immunization Practices (ACIP) recommended routine immunization with three oral doses at 2, 4 and 6 months of age. The tetravalent vaccine (RotaShield) was licensed in the USA by the FDA in August 1998. In July 1999, after about 1.5 million doses had been given, the CDC recommended suspending administration of the vaccine because post-licensure surveillance of adverse events had suggested an association with intussusception. After further investigation by CDC, in October 1999, the ACIP withdrew its recommendation concluding that '…intussusception occurs with significantly increased frequency in the first 1-2 weeks after vaccination with RRV-TV, particularly following the first dose'. The implications of these developments from a practical, epidemiological analytical and ethical perspective are discussed.

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The molecular biology of human caliciviruses Ian N. Clarke and Paul R. Lambden Virus group, Mailpoint 814, Division of Cell and Molecular Medicine, University of Southampton Medical School, Southampton SO16 6YD, UK Within the last decade molecular analyses of the genome of Norwalk-like viruses (NLVs) have confirmed that this important group of infectious agents belong to the Caliciviridae family. NLVs have a positive-sense, single-stranded RNA genome of approximately 7700 nucleotides excluding the polyadenylated tail. The genome encodes three open reading frames: ORF 1 is the largest (~1700 amino acids) and is expressed as a polyprotein precursor that is cleaved by the viral 3C-like protease; ORF 2 encodes the viral capsid (550 amino acids); and ORF 3 encodes a small basic protein of unknown function. Comparative sequencing studies of human caliciviruses have revealed a second distinct group of viruses known as Sapporo-like viruses (SLVs). SLVs also have a single-stranded, positivesense RNA genome of approximately 7400 nucleotides and the small 3¢ terminal ORF (NLV-ORF3 equivalent) is retained. Phylogenetic analyses of NLV and SLV genomic sequences have assigned these viruses to two different genera with each genus comprised of two distinct genogroups. The fundamental difference in genome organization between NLVs and SLVs is that the polyprotein and capsid ORFs are contiguous and fused in SLVs. Progress in understanding the molecular biology of human caliciviruses is hampered by the lack of a cell culture system for virus propagation. Studies on viral replication and virion structure have therefore relied on the expression of recombinant virus proteins in heterologous systems. Norwalk virus capsid expressed in insect cells assembles to form virus-like particles (VLPs). Structural studies have shown that Norwalk virus VLPs are comprised of 90 dimers of the capsid protein.

Molecular epidemiology of human enteric caliciviruses in The Netherlands Marion Koopmans, Jan Vinje, Erwin Duizer, Matty de Wit and Yvonne van Duijnhoven Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment, Bilthoven, The Netherlands Caliciviruses are among the most common causes of gastroenteritis in people of all age groups. These antigenetically and genetically diverse viruses have been grouped into two genera within the family Caliciviridae, designated Norwalk-like (NLV) and Sapporo-like (SLV). To gain more insight in their epidemiology, we have developed a tentative genotyping scheme, which was used to differentiate the viruses detected in a set of epidemiological studies. NLVs and SLVs were detected by generic RT-PCR in stool specimens from 5.1% and 2.4% of cases with acute gastroenteritis for which a general practitioner was consulted, and in 16.5% and 6.3% of community cases of gastroenteritis. In addition, NLVs were associated with more than 80% of reported outbreaks of gastroenteritis from 1994-1999. Typically, several genotypes of NLV co-circulate in the community. Occasionally, however, several consecutive outbreaks were caused by essentially the same virus, although an epidemiological link had not previously been noted. This was most pronounced in 1995/1996, when a Lordsdale-like variant was detected that subsequently was found world-wide. This epidemic spread suggests differences in virulence or mode of transmission. In addition, we found that related NLVs are highly prevalent in calves in The Netherlands, raising questions about their potential for zoonotic transmission.

Molecular biology of astroviruses: selected highlights Suzanne M. Matsui, David Kiang, Nancy Ginzton, Teri Chew and Ute Geigenmüller-Gnirke Department of Medicine, Division of Gastroenterology, Stanford University School of Medicine, Stanford, CA 943055487, and Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA Human astrovirus, the prototype of the Astroviridae family, is a non-enveloped positive-strand RNA virus with distinctive morphology. Initially named for a characteristic 5-6 point star evident on the surface of faecally shed viral particles by direct electron microscopy, a recent study using cryoelectron microscopy and image reconstruction indicates viral particles consist of a smoothly rippled, solid capsid decorated with short spikes. Mechanisms underlying the assembly of these viral particles have not been fully elucidated. However, studies of two full-length clones of human astrovirus serotype 1 suggest that capsid residue Thr227 plays a critical role in the assembly of infectious viral progeny. The development of a full-length clone (pAVIC) from which infectious RNA can be transcribed has also facilitated studies of the viral 3C-like serine protease, encoded in ORF1a. These studies demonstrate that the full-length ORF1a product (101 kDa) is processed in vitro to an N-terminal 64 kDa fragment and a C-terminal 38 kDa fragment. Mutation of the predicted catalytic triad inhibits proteolysis. In other studies based on modifications of pAVIC, preliminary evidence supports the feasibility of developing a reporter cell line to facilitate astrovirus detection.

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Molecular epidemiology of human astroviruses Stephan S. Monroe*, Jennifer L. Holmes*† and Gaël M. Belliot*‡ *Viral Gastroenteritis Section, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA, †Oak Ridge Institute for Science and Education, Oak Ridge, TN, USA, and ‡Atlanta Research and Education Foundation, Atlanta, GA, 30033 USA Human astroviruses (HAstVs) are associated with 5-9 percent of cases of gastroenteritis in young children. Seven serotypes (HastV-1 to -7), which correlate with genotypes, have been defined by using immune typing methods. We have used partial nucleotide sequence information from the capsid protein gene for molecular typing of 29 unique human astrovirus strains obtained from prospective studies of children with gastroenteritis in Egypt and Malawi. HAstV-1 was the most commonly detected strain, consistent with previous studies, but a surprising variety of strains were identified in both collections. An eighth astrovirus type, HAstV-8, has been defined on the basis of complete capsid protein gene sequence and was detected in both collections analysed in this study. Although HAstV-8 and HAstV-4 strains segregate into well resolved clades by analysis of sequences from the region encoding protein P2 (VP32), the pair-wise distances between these types are less than those between strains of the other serotypes. In contrast, analysis of sequences from the region encoding protein P3 unambiguously resolve HAstV-4 and HAstV-8 strains, consistent with their classification as distinct serotypes. Overall, strains representing six of the eight serotypes were detected in two collections of samples from prospective studies of gastroenteritis in young children indicating that multiple astrovirus types are frequently co-circulating within communities.

Enteric infections with coronavirses and toroviruses Kathryn V. Holmes Department of Microbiology, B-175, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, CO 20862, USA Many enteric viruses are difficult or impossible to propagate in tissue culture. Coronaviruses and toroviruses are large, enveloped, plus-strand RNA viruses in the order Nidovirales that cause enteric disease in young pigs, cows, dogs, mice, cats and horses. Two different serogroups of mammalian coronaviruses cause frequent respiratory infections in humans, and coronaviruses and toroviruses have been implicated in human diarrhoeal disease by immunoelectron microscopy. However, there is as yet no consensus about the importance of these enveloped viruses in human diarrhoea, and little is known about their genetic variability. The large spike (S) glycoprotein is an important determinant of species specificity, tissue tropism and virulence of coronavirus infection. To infect enterocytes, both S glycoproteins and the viral envelope must resist degradation by proteases, low and high pH, and bile salts. One specific site on the S glycoprotein of bovine coronavirus must be cleaved by an intracellular protease or trypsin to activate viral infectivity and cell fusion. S glycoprotein binds to specific receptors on the apical membranes of enterocytes, and can undergo a temperature-dependent, receptor-mediated conformational change that leads to fusion of the viral envelope with host membranes to initiate infection. Analysing spike-receptor interactions may lead to new ways to propagate these enteric viruses as well as new strategies for development of novel antiviral drugs.

Viruses causing diarrhoea in AIDS Richard C. G. Pollok Digestive Disease Research Centre, St Bartholomews & The Royal London School of Medicine and Dentistry, Turner St, London E1 2AD, UK Opportunistic viral enteritis is an important gastrointestinal manifestation of HIV related disease. Cytomegalovirus (CMV) and human herpes simplex virus are well established aetiological agents of disease in the gastrointestinal tract in this group. CMV enteritis may affect any region of the bowel, most commonly the colon. Diagnosis and management of these infections may be difficult. The role of other viruses in so called 'pathogen-negative' diarrhoea remains controversial. The clinical importance of HIV-enteropathy is probably limited. Several viruses including astrovirus, picobirnavirus, small round structured virus and rotavirus have been implicated HIV-related diarrhoea. In addition, adenovirus has been linked to persistent diarrhoea in patients with a characteristic adenovirus colitis. The spectrum of disease morbidity and mortality amongst HIV patients has altered dramatically since the wide spread introduction of highly active antiretroviral therapy (HAART). Opportunistic infections, including CMV infection of the gastrointestinal tract in patients with AIDS, have diminished greatly. AIDS patients with CMV are able successfully to discontinue anti-CMV treatment without disease reactivation and with a parallel reduction in CMV viraemia following the initiation of HAART.

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Treatment of gastrointestinal viruses Michael J. G. Farthing Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine & Dentistry, Turner Street, London E1 2AD, UK The most common enteric viruses responsible for diarrhoea are rotavirus, enteric adenoviruses, caliciviruses including the Norwalk agent and astrovirus. These infections are usually mild to moderate in severity, self-limiting and of short duration and thus, specific antiviral therapy is not recommended. The standard management of these infections is restoration of fluid and electrolyte balance and then maintenance of hydration until the infection resolves. WHO oral rehydration therapy (ORT) was introduced about 30 years ago and has saved the lives of many infants and young children. During the last 10 years it has become evident that the efficacy of ORT can be increased by reducing the osmolality of the WHO oral rehydration solution (ORS) to produce a relatively hypotonic solution. Hypotonic ORS appears to be safe and effective in all forms of acute diarrhoea in childhood. Complex substrate ORS, which is also usually hypotonic, has been shown to have increased efficacy in cholera but not in other bacterial or viral diarrhoeas. Nevertheless, the scientific rationale for using rice or resistant starch as substrate in ORS is of physiological interest. Other treatments such as hyperimmune bovine colostrum, probiotics and antiviral agents are largely experimental and have not been introduced into routine clinical practice. Cytomegalovirus (CMV) infection of the gastrointestinal tract occurs mainly in the immunocompromised although it has been reported in immunocompetent individuals. CMV infects both the oesophagus and colon to produce oesophagitis, often with discrete ulcers, and colitis, respectively. Both conditions can be treated with ganciclovir or foscarnet. Failure to respond to monotherapy is an indication to use both agents concurrently.

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