Global study of viral diarrhea in hospitalized children in Spain: results of structural surveillance of viral gastroenteritis net work (VIGESS-net) 2006-2008.

BACKGROUND AND OBJECTIVES: Detection and characterization of gastroenteritis cases (viruses) was conducted during eleven years through the surveillance VIGESS-net, which was created in an effort to conduct a structured surveillance of rotavirus genotypes co-circulating in Spain. STUDY DESIGN AND RESULTS: This phase includes the study of 2048 fecal samples from children <5 years old, hospitalized in fifteen different hospitals throughout Spain from March 2006 to March 2008. Of them, 821 (40.1%) samples were rotavirus positive. Rotavirus was identified as the only etiological agent in 694 (33.9%) cases and in 127 (6.2%) was found as coinfection with other enteric viruses, mainly with noroviruses. Predominant G genotypes detected were G1 (49.8%) followed by G9 (32.9%), G3 (2.6%), G8 (1.0%), G4 (0.4%), G6 (0.2%) and G12 (0.2%). The G2 was encountered in 3.2% of cases. Rotavirus mixed G-types infections occurred in 3.9% of cases. The main G/P combinations were G1P[8] (51.9%) and G9P[8] (35.6%), which predominates alternatively in the first and second period of the study. More rare combinations occur in less than 7.4% of cases. CONCLUSION: The diversity of rotavirus circulating strains suggests to maintain a surveillance system through different regions of the country.

Gastroenteritis vírica en niños hospitalizados. Estudio prospectivo multicéntrico / Viral gastroenteritis in hospitalized children. A multicentre prospective study

Introducción: El objetivo de este estudio fue determinar prospectivamente la etiología vírica de la gastroenteritis aguda (GEA) en lactantes hospitalizados, así como describir las características clínicas de los principales virus. Pacientes y métodos: Desde octubre de 2006 hasta marzo de2007, se realizó el seguimiento de todos los niños de 1-23 meses de edad hospitalizados en tres hospitales, desde el momento de la admisión hasta las 72 horas tras el alta, para detectarla presencia de GEA. Se analizó una muestra de heces de los niños con GEA para la detección de rotavirus, calicivirus (norovirus y sapovirus), astrovirus y adenovirus mediante transcripción inversa y reacción en cadena de la polimerasa. Resultados: De un total de 1.576 pacientes hospitalizados, se pudo realizar el seguimiento de 1.300 (82,5%), que han constituido nuestra cohorte de estudio. Un total de 242 niños tuvieron GEA (un 18,6% de la cohorte), obteniéndose muestra de heces de 217 (89,7%). En 91 casos (42%) se detectaron rotavirus, en 72 norovirus (33,2%), en 7 astrovirus (3,2%) y en2 adenovirus; no se detectó sapovirus en ningún caso. Trece niños (6%) presentaron infecciones mixtas por dos o más virus, y 32 (14,7%) resultaron negativos para todos los virus analizados. En los casos de GEA por norovirus se observa menor apatía y pérdida de peso que en los casos de rotavirus, pero mayorporcentaje de heces con sangre.Conclusiones: Los virus son una causa frecuente de GEA enlos niños menores de 2 años hospitalizados; el principal virus hallado es el rotavirus, seguido del norovirus. Debido a las escasas diferencias clínicas observadas entre rotavirus y norovirus, es necesaria su determinación analítica para su diferenciación (AU)

Introduction: The objective of this study was to determine prospectively the viral etiology of acute gastroenteritis (AGE) in hospitalized weaning babies and describe the clinical characteristics of the principal virus detected. Patients and methods: All children aged 1 to 23 months admitted to 3 hospitals during October 2006-March 2007 were followed for the presence of AGE from the time of hospital admission until 72 hours after hospital discharge. A stool sample of children with AGE was tested for calicivirus (norovirus and sapovirus), rotavirus, adenovirus and astrovirus by RT-PCR. Results: Of the 1,576 hospitalized children, 1,300 (82.5%)were fully monitored being our study cohort. A total of 242children had AGE (18.6% of the cohort) and stool samples from217 children were obtained (89.7%). In 91 cases (42%) were positive to rotavirus, 72 (33.2%) to norovirus, 7 (3.2%) to astrovirus, and in 2 to adenovirus, not detecting sapovirus in any case, 13 children (6%) showed mixed infections by two or more viruses and 32 (14.7%) cases were negative for the analyzed virus. Norovirus AGE have less apathy and weight loss than thecases of rotavirus but a higher percentage of stools with blood. Conclusions: The viruses are a frequent cause of AGE among the hospitalized children of less than 2 years of age being rotavirus the principal virus found followed by norovirus. Few clinical differences were observed between rotavirus and norovirus and analytical determination is necessary for their differentiation (AU)

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network.

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Rotavirus surveillance in europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data.

BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

Identification of risk factors associated with nosocomial infection by rotavirus P4G2, in a neonatal unit of a tertiary-care hospital.

A rotavirus outbreak in newborns admitted to the 'La Paz' University Hospital, Madrid was detected, followed up and controlled. Uninfected children were selected as control subjects. Samples of faeces were taken once or twice weekly from all the newborns, including those who were asymptomatic and who were admitted to the neonatal unit for early detection of rotavirus and the positive were separated from the rest of the neonates. Contact-related precautions were taken for all patients, and alcohol solutions were used for hand washing. During the months of the outbreak, 1773 children were admitted to the hospital, 131 of whom were affected by the rotavirus infection (7.4%). Of these, 72 (55%) had symptomatic infections. In the first month of the outbreak, nine cases of necrotizing enterocolitis were diagnosed (one patient developed massive intestinal necrosis). The infections (symptomatic and asymptomatic) presented a bimodal distribution caused by a new outbreak of rotavirus type P4G2 after two patients who had acquired the infection outside the hospital were admitted when the first outbreak was subsiding. The characteristics of cases and controls were analysed using bivariate and multivariate methods (non-conditional multivariate logistic regression) to identify four risk factors strongly associated with rotavirus infection: premature birth, infections other than rotavirus, malformation, and changes in glycaemia and/or presence of jaundice.

Evaluation of two commercial enzyme immunoassays for the detection of norovirus in faecal samples from hospitalised children with sporadic acute gastroenteritis.

Two commercially available enzyme immunoassays (EIAs), IDEIA and Ridascreen, for norovirus antigen detection were evaluated with 117 faecal samples from hospitalised children with acute gastroenteritis. Eighteen of 39 samples positive by RT-PCR were characterised by sequence analysis, and 17 of these were related to norovirus genogroup II. When compared with RT-PCR, the sensitivity and specificity values were 76.9% and 85.9%, respectively, for the IDEIA assay, and 59.0% and 73.1%, respectively, for the Ridascreen assay. The sensitivity and specificity of both EIA tests require improvement, but they could both eventually be of use in the diagnosis of norovirus diarrhoea in clinical laboratories.

Rotavirus G9 es la principal causa de diarrea aguda en niños ingresados menores de 5 años / Rotavirus G9 is the principal cause of acute diarrhea in hospitalized children under five years of age

Objetivos: analizar el perfil etiológico de la diarrea aguda en niños ingresados menores de 5 años, y la distribución de los serotipos de rotavirus en nuestro medio. Métodos: estudio prospectivo observacional descriptivo llevado a cabo en todos los niños de 1 mes a 5 años de edad, ingresados por diarrea aguda en el Servicio de Pediatría del Hospital de León, entre 15 de enero y 31 de diciembre de 2005. En todos los casos se realizó coprocultivo para bacterias enteropatógenas, y estudio de virus en heces por inmunocromatografía para rotavirus y adenovirus, y enzimoinmunoensayo para astrovirus, y posteriormente por reacción en cadena de la polimerasa a tiempo real (RT-PCR) para rotavirus, analizando con esta última los genotipos G del mismo. Idéntico estudio se llevó a cabo en los casos de diarrea nosocomial. Resultados: en el período estudiado hubo un total de 1.037 ingresos (excluída la edad neonatal), de los cuales 674 (65%) fueron menores de 5 años. Se incluyeron en el estudio 106 niños: 90 ingresados por diarrea aguda comunitaria (13,3 % de los ingresos en ese grupo de edad), y 16 casos de diarrea nosocomial. En el primer grupo se aisló un patógeno bacteriano en un 13,3% (Campylobacter y Salmonella a partes iguales), y en el 43,3% un virus (rotavirus 87,1%, astrovirus 17,9%, y adenovirus 5,1%), con algunos casos de coinfección. Rotavirus se aisló por tanto en un 37,7% de los ingresos por diarrea comunitaria. El único gérmen aislado en la diarrea nosocomial fue rotavirus, en un 37,5%. Se analizó el tipo G de 26 cepas de rotavirus: G9 57,7%, G1 15,4%, G2 11,5%, G3 11,5%, G4 0, y G9+G3 3,8%. Conclusiones: rotavirus es la causa más frecuente de diarrea aguda en niños hospitalizados menores de 5 años, siendo responsable de más de un tercio de los casos tanto en la diarrea comunitaria como en la nosocomial. Más de la mitad de los rotavirus aislados mostraban el tipo G9

Objectives: analyze the etiological profile of acute diarrhea in hospitalized children under five years of age and the distribution of the rotavirus serotypes in our setting. Methods: perspective, observational, descriptive study conducted in all the children from one month to five years of age hospitalized due to acute diarrhea in the Pediatrics Department of the Hospital de Leon, between January 15 and December 31, 2005. In all the cases, stool culture was made for enteropathogenic bacteria and the virus study was done in stools by immunochromatography for rotavirus and adrenovirus and enzyme immunoassay was done for astrovirus and subsequently by real time polymerase chain reaction (RT-PCR) for rotavirus, analyzing the G genotypes in the latter. An identical study was conducted in the cases of nosocomial diarrhea. Results: in the period studied, there were a total of 1037 hospital admissions (neonatal age being excluded). Atotal of 674 (65%) of them were under five years of age. One hundred six children were included in the study: 90 admitted for community acute diarrhea (13.3% of those admitted in this age group), and 16 cases of nosocomial diarrhea. In the first group, a bacterial pathogen was isolated in 13.3% (Campylobacter and Salmonella equally). In 43.3%, a virus (rotavirus 87.1%, astrovirus 17.9%, and adenovirus 5.1%) was isolated with some cases of coinfection. Thus, rotavirus was isolated in 37.7% of those admitted due to community diarrhea. The only germ isolated in nosocomial diarrhea was rotavirus in 37.5%. The type G of 26 rotavirus strains was analyzed: G9 57.7%, G1 15.4%, G2 11.5%, G3 11.5%, G4 0, and G9+G3 3.8%. Conclusions: rotavirus is the most frequent cause of acute diarrhea in hospitalized children under five years of age, it being responsible for more than one third of the cases of both community and nosocomial diarrhea. More than half of the rotaviruses isolated had type G9

Diagnóstico mediante ELISA de un brote de infección gastrointestinal por Norovirus en una guardería infantil / ELISA Diagnosis of a gastrointestinal norovirus outbreak in a nursery

La incidencia de brotes de gastroenteritis por Norovirus en niños ha sido infravalorada. Esto se ha debido principalmente a la dificultad para disponer de procedimientos diagnósticos adecuados (microscopía electrónica y reacción en cadena de polimerasa de trascripción inversa[RT-PCR]). Los nuevos métodos de enzimoinmuno ensayo (ELISA), capaces de detectar antígenos virales en heces, constituyen una alternativa diagnóstica prometedora. El objetivo de este estudio es describir la aplicación de una técnica de ELISA en la investigación de un brote de gastroenteritis por Norovirus en una guardería. En septiembre de 2003 se notificaron varios casos de gastroenteritis entre niños que acudían a una guardería. El brote afectó a 15 de los 64 niños expuestos. La sintomatología fue vómitos (100%), dolor abdominal (66%), náuseas (40%) y diarrea (40%). La duración del cuadro fue de 3 días (mediana 2 días). Tres pacientes requirieron ingreso hospitalario y todos se recuperaron con tratamiento sintomático. Se estudiaron 3 muestras de heces de 3 pacientes. La presencia de Salmonella sp, Shigella sp y Campylobacter sp se descartó por coprocultivo. La investigación de Norovirus, Rotavirus, Adenovirus y Astrovirus se realizó mediante ELISA específicos (IDEIA NLV, IDEIA Rotavirus, IDEIA Adenovirus e IDEIA Astrovirus; Dako Ltd, Ely UK). En las 3 muestras procesadas se obtuvieron resultados positivos por ELISA para Norovirus y negativos para el resto de los patógenos. La confirmación de resultados para Norovirus se llevó a cabo mediante RT-PCR. La aplicación generalizada de técnicas de ELISA permitirá ayudar a definir en nuestro entorno el papel de los Norovirus como agentes responsables de brotes de gastroenteritis aguda en niños

The incidence of gastrointestinal outbreaks caused by Norovirus in children has been underestimated. This fact is mainly due to the difficulty in the availability of adequate diagnostic procedures (electronic microscopy and Reverse Transcriptase Polymerase Chain Reaction [RTPCR]).The new Enzyme-Linked Immunosorbent Assay (ELISA) methods for the detection of viral antigens in stools represent a promising diagnostic alternative. The objective of this study was to describe the application of an ELISA technique in the investigation of an outbreak by Norovirus in a day care center. In September 2003 several cases of gastroenteritis in children attending a day care center were communicated. The outbreak affected to 15 out of 64 exposed children. The symptomatology was vomits (100%), abdominal pain (66%), nauseas(40%) and diarrhoea (40%). The duration of the symptoms was 3 days (median 2 days). Three patients required hospitalisation and all of them improved with symptomatic treatment. Three faecal samples of three patients were studied. The presence of Salmonella sp, Shigella sp and Campylobacter sp was discarded by coproculture. The investigation of Norovirus, Rotavirus, Adenovirus and Astrovirus was carried out by specific ELISAs (IDEIA NLV, IDEIA Rotavirus, IDEIA Adenovirus e IDEIA Astrovirus; Dako Ltd, Ely UK). In the three processed samples were obtained positive results for Norovirus. These results were confirmed by using RT-PCR. The results for all other pathogens were negative. The wide application of similar ELISA techniques will allow defining the role of Norovirus as agents of gastrointestinal outbreaks among children in our environment

Gastroenteritis aguda nosocomial e infección asintomática por rotavirus y astrovirus en niños hospitalizados / Nosocomial gastroenteritis and asymptomatic rotavirus and astrovirus infection in hospitalized children

Antecedentes: La gastroenteritis nosocomial es un problema frecuente en las unidades de hospitalización pediátricas, donde el 20-50 por ciento de los episodios por rotavirus y astrovirus son de origen nosocomial. Objetivo: Describir en nuestro medio la incidencia de gastroenteritis nosocomial por rotavirus y astrovirus y de eliminación fecal asintomática de estos virus e identificar los serotipos G de los rotavirus detectados. Métodos: Estudio prospectivo de los niños ingresados durante un año en la unidad de lactantes con seguimiento de la aparición de síntomas de gastroenteritis y estudio periódico de heces para detección de antígeno de rotavirus y de astrovirus por enzimoinmunoanálisis (EIA). En los pacientes con gastroenteritis se realizó también coprocultivo, estudio de adenovirus por EIA y de calicivirus por reacción en cadena de la polimerasa (PCR) y análisis de los serotipos G de los rotavirus por EIA con anticuerpos monoclonales. Resultados: De los 666 niños ingresados sin diarrea, 60 presentaron gastroenteritis nosocomial (9/100 pacientes ingresados y 1,75 por 100 días de estancia hospitalaria): rotavirus en 34 casos (5/100 pacientes) y astrovirus en 2 (0,3/100 pacientes). En 27 pacientes de los 329 estudiados sin diarrea se detectó eliminación viral: rotavirus en 23 pacientes y astrovirus en cuatro; en 13 (4 por ciento) en el ingreso y en 14 (4,2 por ciento) pasadas 72 h (infección nosocomial asintomática). No hubo diferencia en los serotipos G detectados entre los casos de gastroenteritis nosocomial y los de origen comunitario. Conclusiones: Se confirma la importancia de la etiología viral en la gastroenteritis nosocomial y se valora la eliminación fecal asintomática de rotavirus como uno de los factores de transmisión de esta infección (AU)

[Nosocomial gastroenteritis and asymptomatic rotavirus and astrovirus infection in hospitalized children]. / Gastroenteritis aguda nosocomial e infección asintomática por rotavirus y astrovirus en niños hospitalizados.

BACKGROUND: Nosocomial gastroenteritis is frequent in pediatric hospital wards. Between 20% and 50% of gastroenteritis cases caused by rotavirus and astrovirus are of nosocomial origin. OBJECTIVE: To determine the incidence of nosocomial rotavirus and astrovirus gastroenteritis in our environment, the incidence of asymptomatic infection with these viruses, and to identify the G serotypes of the rotaviruses detected. METHODS: We performed a prospective study of all children under 2 years of age admitted to a neonatology unit over a 1-year period who were followed-up for the presence of diarrhea and periodic study of feces to detect the presence of rotavirus and astrovirus antigens by enzyme immunoassay (EIA). Patients with gastroenteritis also underwent bacteria stool culture, adenovirus detection by EIA, calcivirus detection by polymerase chain reaction, and analysis of rotavirus G serotypes by EIA with monoclonal antibodies. RESULTS: Of 666 children admitted without diarrhea, 60 presented nosocomial gastroenteritis (9 % of patients admitted and 1.75 per 100 days of hospital stay): 34 presented rotavirus (5 % of patients) and two presented astrovirus (0.3 % of patients). Of the 329 patients without diarrhea who were studied, viral elimination was detected in 27: rotavirus in 23 patients and astrovirus in four. Viral infection was detected on admission in 13 patients (4 %) and after 72 hours in 14 patients (4.2 %) (asymptomatic nosocomial infection). No differences in the distribution of rotavirus G serotypes were observed between community-acquired and nosocomial gastroenteritis. CONCLUSIONS: These data confirm the importance of viral etiology in nosocomial gastroenteritis and allow us to evaluate asymptomatic fecal elimination of rotavirus as one of the factors in the transmission of this infection.

Viruses causing gastroenteritis.

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viruses are recognized as important causes of this disease, particularly in children. Since the Norwalk virus was identified as a cause of gastroenteritis, the number of viral agents associated with diarrheal disease in humans has steadily increased. Rotavirus is the most common cause of severe diarrhea in children under 5 years of age. Astrovirus, calicivirus and enteric adenovirus are also important etiologic agents of acute gastroenteritis. Other viruses, such as toroviruses, coronaviruses, picobirnaviruses and pestiviruses, are increasingly being identified as causative agents of diarrhea. In recent years, the availability of diagnostic tests, mainly immunoassays or molecular biology techniques, has increased our understanding of this group of viruses. The future development of a safe and highly effective vaccine against rotavirus could prevent, at least, cases of severe diarrhea and reduce mortality from this disease.

First detection of group C rotavirus in children with acute diarrhea in Spain.

Group C rotavirus causes sporadic cases and outbreaks of acute diarrhea in children and adults in many countries, but has never been detected among children in Spain. In a recently conducted surveillance study to screen fecal specimens for bacteria and viruses from a cohort of 822 young children who were treated for acute diarrhea in Madrid, no pathogens were detected in fecal specimens from 238 (29%) children. In this study, we examined 147 of those specimens for group C rotavirus by EIA and PCR and found 22 (15%) were positive. Our findings demonstrate that group C rotavirus is an important cause of childhood diarrhea in Spain.

Molecular detection of human calicivirus among Spanish children with acute gastroenteritis.

A survey was conducted among Spanish children with gastroenteritis treated in an emergency room. Reverse transcription-PCR with specimens negative for other enteric pathogens was used. The minimum incidence of human calicivirus infection was 7.7%, with Lordsdale as the predominant genotype. The clinical features and severity of calicivirus and rotavirus were similar.

New immunochromatographic method for rapid detection of rotaviruses in stool samples compared with standard enzyme immunoassay and latex agglutination techniques.

Three different commercial immunologic tests for rapid detection of group A rotavirus (an immunochromatographic method, latex agglutination, and enzyme immunoassay) were used to evaluate 228 faecal specimens obtained from Spanish children with acute gastroenteritis. After resolution of 30 (13.2%) discordant results by reverse transcription-polymerase chain reaction for rotavirus, the statistical values of the enzyme immunoassay, latex agglutination, and immunochromatographic method were respectively 96%, 68%, and 99% for sensitivity; 99%, 99%, and 96% for specificity; 98%, 96%, and 92% for positive predictive value; and 98%, 88%, and 99% for negative predictive value. The immunochromatographic technique showed high sensitivity and specificity and was rapid and easy to perform in the routine clinical laboratory.

[The molecular epidemiology of the rotavirus in Spanish children. The Rotavirus Study Group (GER)]. / Epidemiología molecular de rotavirus en niños españoles. Grupo de Estudio de Rotavirus (GER).

BACKGROUND: Rotavirus are the most common etiologic agent of acute gastroenteritis in childhood. The knowledge of the circulating antigenic types is important in development of future vacunes. METHODS: Faeces from children (age < 4 years) with acute gastroenteritis admitted in the two hospitals (Hospital Severo Ochoa-Madrid and Hospital General Vic-Barcelona) have been studied prospectively during one year (October-1996 to October-1997). The detection of rotavirus was performed by ELISA (IDEIA, Dako). All samples were G-serotyping by EIA-Mabs (Silenius Laboratories) and the indeterminate or non-serotypable samples were G-genotyping by RT-PCR. P genotypes were identified by RT-PCR. RESULTS: 322 (45%) patients with acute diarrhoea causing for rotavirus were confirmed, 242 coming from the Madrid metropolitan area and other 80 from the Barcelona area. The EIA-Mabs technique made it possible to identify the G serotypes in 287 cases (89%), corresponding 207 to G1 serotype, 70 to G4 serotype and 6 to G3 serotype. In 4 patients both G1 and G4 serotypes were detected. The EIA-Mabs could not determined the serotype in 35 (11%) patients, all of whom were confirmed by RT-PCR (12 belonged to serotype G1 and 23 to serotype G4). Analysis of P genotypes was carried out in 25 patients obtained from Madrid and 17 from Barcelona; all cases were classified in the P[8] genotype. CONCLUSIONS: The most frequent serotype in both hospitals was G1. The EIA-Mabs technique were showed a high sensitivity, however, the RT-PCR technique used were even more efficient, making it possible for us to identify all the non-serotypable EIA-Mabs cases. The temporal study of circulating serotypes/genotypes of rotavirus is necessary to evaluate the efficiency of vaccines.

Ultrastructure of human astrovirus serotype 2.

The ultrastructure of human astrovirus serotype 2 (H-Ast2) grown in cell culture was analysed by electron microscopy of thin sections and negatively stained preparation. Infected LLCMK2 cells, as visualized in thin sections, contained cytoplasmic aggregates of dense or hollow-cored particles that aggregated in quasicrystalline arrays and were specifically labelled using a rabbit polyclonal anti-Ast2 antiserum. H-Ast2 particles from the supernatant of infected LLCMK2 cells in thin sections after flat- embedding were similar in size to intracellular virions. In negatively stained preparations, these virus particles had an external diameter of 41 nm and exhibited a well defined layer of surface spikes. Pentagonal and hexagonal contours were occasionally visible, and probably correspond to the projections of icosahedral structures. Star-like morphologies and particles with surface triangular hollows were seen in dark areas of the preparations only after a short treatment of the viruses of pH 10. Incubation of the viruses at pH 10.5 induced a rapid disassembly of the virus particles. The finding that the particles with icosahedral geometry and surface spikes are fully infective allows an alternative morphological model to the traditional one for astroviruses to be proposed.

Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.

Characterization of a human astrovirus serotype 2 structural protein (VP26) that contains an epitope involved in virus neutralization.

An improved purification procedure for human astrovirus serotype 2 (H-Ast2) has facilitated the isolation of a neutralizing monoclonal antibody (PL-2) directed against one of the major structural proteins (VP26) of H-Ast2. A minor component (VP29) of the virus particles is also recognized by PL-2 antibody. Immunofluorescent staining indicated that VP26 (and/or VP29) has mainly a cytoplasmic location in LLCMK2-infected cells. Immunoelectron microscopy demonstrated that the PL-2 epitope was present in the surface of astrovirus particles. Pulse-chase radiolabeling and immunoprecipitation of H-Ast2-infected cell extracts identified a P86 precursor of VP26. Several intermediate protein species (P74 to P35) that shared the PL-2 epitope were also identified in the infected cells. Finally, partial N-terminal sequencing of VP29 and VP26 polypeptides demonstrated that they originated by alternative processing of P86 after residues 361 and 394, respectively. These results corroborate the location of the astrovirus structural genes at the 3' end of the viral genome included in the previously identified 2.8-kb subgenomic RNA.

Conservation of epitopes recognized by monoclonal antibodies against the separated subunits of influenza hemagglutinin among type A viruses of the same and different subtypes.

Monoclonal antibodies raised against the separated hemagglutinin subunits (HA1 and HA2) of influenza A/Vic/3/75 (H3N2) virus were tested against a large panel of human and avian strains. The epitopes recognized by most antibodies were conserved among subtype H3 viruses, but reactivity of some antibodies with members of other subtypes was also observed. Particularly, the H4 virus reacted with most antibodies directed against the HA2 subunit. These results are discussed in terms of sequence similarities between subtypes and application of these antibodies as subtyping reagents.

Isolation of cross-reactive, subtype-specific monoclonal antibodies against influenza virus HA1 and HA2 hemagglutinin subunits.

The large (HA1) and small (HA2) subunits of influenza virus A/Vict/3/75 hemagglutinin were purified in denatured form by preparative electrophoresis. Both polypeptides were used to immunize mice from which monoclonal antibodies were obtained. These antibodies reacted not only with the corresponding hemagglutinin subunit but also with purified virions. When tested by radioimmunoassay against a panel of human viruses, most anti-HA1 and -HA2 antibodies behaved as subtype-specific, whereas anti-HA antibodies, raised against purified virus, were more restricted. The anti-subunit antibodies were negative in hemagglutination-inhibition and neutralization tests. The interest of these antibodies as reagents for research and diagnosis is discussed.