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OBJECTIVE To evaluate long-term global and site-specific health-related quality of life (HRQoL) in patients treated for sinonasal and nasopharyngeal malignancies. STUDY DESIGN Cross-sectional. METHODS One hundred fourteen patients with sinonasal and nasopharyngeal malignancies received surgery, radiation, systemic chemotherapy, or a combination thereof, with curative intent. Validated global ([EuroQol-5D] Visual Analogue Scale [EQ-5D VAS]) and disease-specific instruments (MD Anderson Symptom Inventory-Head and Neck [MDASI-HN], Anterior Skull Base Questionnaire [ASBQ]) were administered to patients who were both free of disease and had completed treatment at least 12 months previously. Associations between instruments, instrument domains, and specific clinical parameters were analyzed. RESULTS The median age was 55 years. The mean EQ-5D VAS, MDASI-22 composite score, and ASBQ score were 74 (standard deviation [SD] 21), 48 (SD 36), and 130 (SD 27), respectively. The most frequently reported high-severity items in MDASI-HN were dry mouth and difficulty tasting food. The most frequently reported high-severity items in ASBQ were difficulty with smell and nasal secretions. Advanced Tumor (T) classification was associated with worse overall ASBQ sum score (P = 0.02). ASBQ performance at home and MDASI-HN drowsy symptom items independently predicted worse global HRQoL as measured by the EQ-5D VAS (P

To identify a clinically meaningful cut-point for the single item dry mouth question of the MD Anderson Symptom Inventory-Head and Neck module (MDASI-HN).Head and neck cancer survivors who had received radiation therapy (RT) completed the MDASI-HN, the University of Michigan Hospital Xerostomia Questionnaire (XQ), and the health visual analog scale (VAS) of the EuroQol Five Dimension Questionnaire (EQ-5D). The Bayesian information criteria (BIC) were used to test the prediction power of each tool for EQ-5D VAS. The modified Breiman recursive partitioning analysis (RPA) was used to identify a cut point of the MDASI-HN dry mouth score (MDASI-HN-DM) with EQ-5D VAS, using a ROC-based approach; regression analysis was used to confirm the threshold effect size.Two-hundred seven respondents formed the cohort. Median follow-up from the end of RT to questionnaire completion was 88 months. The single item MDASI-HN-DM score showed a linear relationship with the XQ composite score (ρ = 0.80, p 0.001). The MDASI-HN-DM displayed improved model performance for association with EQ-5D VAS as compared to XQ (BIC of 1803.7 vs. 2016.9, respectively). RPA showed that an MDASI-HN-DM score of ≥6 correlated with EQ-5D VAS decline (LogWorth 5.5).The single item MDASI-HN-DM correlated with the multi-item XQ and performed favorably in the prediction of QOL. A MDASI-HN-DM cut point of ≥6 correlated with decline in QOL.

The sodium salts of 2-difluoromethyl-phenyl-alpha-ketoside of N-acetyl-neuraminic acid (compound 1) and of 4-difluoromethyl-2-methoxy-phenyl-alpha-ketoside of N-acetylneuraminic acid (compound 2) were designed as potential mechanism-based inhibitors of sialidase. In vitro both of these compounds competitively inhibited the sialidases of Clostridium perfringens and of influenza virus A/HK/1/68. Inhibition was irreversible with the sialidase of Clostridium perfringens whereas it was reversible with that of A/HK/1/68. Compound 2 did not inhibit the hemagglutinin of the virus but exhibited significant anti-influenza activity when added to the medium of Madin-Darby canine kidney (MDCK) cells infected by influenza virus. In non-infected MDCK cells no inhibition of cellular sialidase was observed. Compound 2 did not block primary infection, but inhibited the release of progeny virus from infected cells. Even after 8 passages in its presence, no resistant strains were detected. Because of its high Ki (8 x 10(-5) M) compared to the low Ki (1' x 1(-10) M) of 4 guanidino-Neu 5 Ac 2en and its reversible inhibition of viral sialidase, its development as an anti-influenza agent is no longer envisaged. Nevertheless, as a mechanism-based irreversible inhibitor of the bacterial enzyme, it could at least be useful for investigating the intrinsic role of sialidase in infections caused by this strain.

Cancers arising from the oropharynx have become increasingly more studied in the past few years, as they are now epidemic domestically. These tumors are treated with definitive (chemo)radiotherapy, and have local recurrence as a primary mode of clinical failure. Recent data suggest that ‘radiomics’, or extraction of image texture analysis to generate mineable quantitative data from medical images, can reflect phenotypes for various cancers. Several groups have shown that developed radiomic signatures, in head and neck cancers, can be correlated with survival outcomes. This data descriptor defines a repository for head and neck radiomic challenges, executed via a Kaggle in Class platform, in partnership with the MICCAI society 2016 annual meeting.These public challenges were designed to leverage radiomics and/or machine learning workflows to discriminate HPV phenotype in one challenge (HPV status challenge) and to identify patients who will develop a local recurrence in the primary tumor volume in the second one (Local recurrence prediction challenge) in a segmented, clinically curated anonymized oropharyngeal cancer (OPC) data set.

The relationship between oxidative stress and neuronal cell death has been suggested for many years. To understand the influence of oxidative stress on neuronal cell death, we investigated the influence of oxidative stress on DEV cells, a human glial cell line. Using enterovirus infection and/or malnutrition to induce oxidative stress, our results demonstrate that those stressors severely influence the antioxidant defense system in DEV cells. Although the expression of mitochondrial manganese superoxide dismutase (MnSOD) in DEV cells was significantly increased in acute infection with viral and nutritional stress, in persistent infection and nutritional stress, the expression of the MnSOD was drastically downregulated. We believe that this downregulation of MnSOD expression in the chronic stress model is due to repression of antioxidant defense. The downregulation of the MnSOD expression may lead to an increase of free-radical production and thus explain why the cells in the chronic stress model were more vulnerable to other oxidative stress influences. The vulnerability of DEV cells to additional stress factors resulted in progressive cell death, which may be analogous to the cell death in neurodegenerative diseases.

Rapid and efficient uptake of glutamate via the high-affinity glutamate transporter EAAT2 is important for limiting glutamate-mediated excitotoxicity involved in neuronal death. Furthermore, there is evidence of altered glutamate uptake and catabolism in motor neuron diseases. Such a defect has been reported in amyotrophic lateral sclerosis, the major motor neuron disease, and was associated with impairment in EAAT2 processing. We recently reported the presence of enterovirus genome specifically in the anterior horn of amyotrophic lateral sclerosis cases, suggesting the involvement of a chronic/persistent enterovirus infection in amyotrophic lateral sclerosis. To investigate a putative link between enterovirus infection and the glutamate-mediated excitotoxicity observed in amyotrophic lateral sclerosis, we developed an in vitro model consisting of a human glial cell line infected with ECHOvirus 6, one of the enteroviruses with sequences closely related to those detected in patients with amyotrophic lateral sclerosis. In these glial cells, an ECHOvirus 6 chronic infection was established, resulting in altered extracellular glutamate uptake. This correlated with an aberrant splicing of the EAAT2 pre-messenger ribonucleic acid and a significant loss of EAAT2 protein expression, similar to that observed in patients with amyotrophic lateral sclerosis. These results provide convincing evidence that an enterovirus chronic/persistent infection may alter glial glutamate uptake and catabolism. As enteroviruses are extremely common human pathogens, they may act as a trigger in the development of certain motor neuron diseases, such as amyotrophic lateral sclerosis.

The Quality Control Assessment (QCA) was initiated to evaluate the quality of the influenza and respiratory syncytial virus (RSV) testing in the national reference centres belonging to the European Influenza Surveillance Scheme (EISS) network. Samples were coded and sent in two panels of 12 samples within a two week interval to 16 laboratories during the 2000-01 winter season. The antibodies titration by HI test was reported by 60% of the laboratories (n=16), and the results were correct for 56% of them. One false detection of influenza B antibodies was reported by one laboratory, and for the others the sensitivity varied widely. The sensitivity of the tests for the detection of influenza virus varied for A(H3N2) from 10 to 100 000 TCID50/ml. The influenza A subtyping was performed by 87% of the laboratories, and 31% gave correct results. The characterisation of the variants was undertaken by six laboratories and half of them fully achieved it. Fifty six percent of the laboratories used RT-PCR for the diagnosis; the results were specific and the sensitivity equivalent to the cell culture.

The efficiency of influenza vaccine was evaluated in the working population by comparing the percentage of people presenting with an influenza-like illness (ILI) according to their influenza immunization status, drug expenses and workdays lost. A self-completed questionnaire about the vaccination was sent to 5785 people randomly chosen among 18 249 workers. When any sick leave was incurred amongst the respondents (63.3%), of whom 301 were vaccinated and 3362 unvaccinated, a clinical form was completed by the private physician and the medical adviser of the firm (Electricité de France and Gaz de France). A final self-completed questionnaire was sent to people whose sick leave was not documented by a physician's reported diagnosis. In total, we obtained complete data for 90.9% of the sampling. The vaccine coverage rate of 8.2% [95% confidence interval (95% CI) = 7.4-9.0%] was higher in men than in women, increasing with age and professional category. Among the 775 subjects with a medical diagnosis, the vaccine effectiveness was not significant: 27.3% (95% CI = -13.8 to 53.5%). In the unvaccinated group, 9.6% had days absent from work, versus 7.0% in the vaccinated group; the two populations were comparable in terms of clinical symptoms, smoking habits, exposure to respiratory risk factors and chronic pathology. The average duration of sick leave for ILI was not significantly different between vaccinated (0.5 days) and unvaccinated workers (0.6 days). Despite the large size of the population and the occurrence of an epidemic due to a virus closely related to the vaccine strain (A/Wuhan/359/95), the vaccine did not effectively protect the small vaccine group nor result in an economic benefit, whatever the professional group.

H3N2 and “avian-like” H1N1 subtypes have circulated in European pigs since the mid- to late 1970s. Following reassortment to acquire six internal genes of the H1N1 viruses in the early 1980s, the H3N2 viruses have evolved to produce antigenically distinguishable variants. Sw/Finistere/127/99, isolated from a pig in January 1999, was shown to be closely related in antigenic and genetic characteristics to contemporary human A/Sydney/5/97-like viruses, and may represent the emergence of another “human-like” virus in European pigs. The H1N1 viruses circulating in pigs in France have evolved gradually over the past 20 years, but are in general closely related to early isolates such as Sw/Finistere/2899/82. An antigenically distinct variant, represented by Sw/Ille et Vilaine/1455/99, represents a significant drift from other recent French H1N1 viruses. Most of the H1N2 reassortant viruses isolated in France and Italy since 1997 were shown to be genetically similar to the H1N2 viruses which have become prevalent in the U.K., but to exhibit significant variation in antigenicity. Genetic reassortment between H1N2 and H1N1 viruses has also contributed to the recent increase in diversity of viruses circulating in pigs in Europe, and emphasizes the potential difficulties in controlling influenza by vaccination. Since most of these swine viruses are resistant to amantadine, they provide an increasing pool of amantadine-resistant M genes for potential incorporation into future human viruses.

We report a case of vaccine-associated paralytic poliomyelitis (VAPP) in Bahrain. The case occurred in an 8-week-old infant who had received a dose of oral polio vaccine (OPV) 7 days after birth. She was in contact with two vaccinees who had received OPV during the national immunisation campaign conducted 10 days before her birth. Specimens from the infant were sent to the WHO Collaborating Centre for Virus Reference and Research Laboratory for serological testing and virus detection, including genomic sequencing. Clinical and virological features are presented of a case of VAPP caused by the Sabin 3 strain of poliovirus that had reverted towards neurovirulence. The case represents one in 51,879 first doses of OPV distributed between 1995 and 1998. In order to reduce further the risk of VAPP, the dose of OPV at birth has been discontinued and a sequential schedule of inactivated polio vaccine (IPV) followed by OPV will be recommended.

Background Twice-daily therapy with famciclovir (FCV) was shown to be effective for episodic therapy for recurrent genital herpes in a large placebo-controlled trial. However, no study has been published to date comparing FCV and aciclovir (ACV). Objectives We have evaluated the effectiveness of FCV vs. ACV in the treatment of recurrent genital herpes infection. Methods A multicentre, double-blind, double-placebo, randomized, parallel-design study, assessed for equivalence, was conducted. As the analysis was based on confidence intervals, a difference of lesion healing time between ACV and FCV (Δ) of 1·05 days with a standard deviation of 2·30 days was chosen. Two hundred and four outpatients were included. Patients self-initiated oral therapy with 125 mg of FCV twice daily or ACV 200 mg five times daily for 5 days. The principal end-point of the study was the complete healing of lesions. Duration of the complete resolution of all symptoms, and safety were also considered. Results The mean healing time was 5·1 days and 5·4 days for FCV and ACV, respectively, with a crude value of Δ = 0·25 days (CI 95%: −0·32; 0·82) in the intent-to-treat population. Therefore, the confidence interval for the difference between the two treatments lies entirely within the equivalence range (−1·05–1·05). The value of Δ in the per-protocol population [0·35 day (CI 95%: −0·24; 0·93)] was comparable between the two groups. No differences were detected in the proportion of patients having complete healing at the different days of evaluation as well as in the duration until the complete resolution of all the symptoms. The frequency, nature and severity of adverse events did not differ among the two treatment groups. Conclusions Twice-daily FCV was as effective and safe in the treatment of recurrent genital herpes simplex virus infection as five times daily ACV.

The pathophysiology of chronic hepatitis in rabbits infected with coxsackievirus B5 (CVB5), (strain Mitchell) was investigated. Three-week-old male New Zealand White rabbits were inoculated intraperitoneally with 1 x 10(5) plaque forming units of virus. Every 3 months for 15 months postinoculation (p.i.) groups of animals were sacrificed for the following tests: interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-beta cytokine levels by enzyme-linked immunosorbent assay (ELISA); splenic natural killer (NK) cell function; sequence of a 154-bp section of the 5' noncoding region; antihepatocyte autoantibodies; histologic examination; in situ polymerase chain reaction (ISPCR) of the liver; neutralizing antibody response to CVB5; and viral cultures of liver, spleen, blood, brain, heart, skeletal muscle, and pancreas samples. Histologic evidence of hepatocyte necrosis was evident at each time point, although few inflammatory cells were seen. Liver samples were positive at each time by ISPCR, with viral nucleic acid localized to hepatocyte cytoplasm. Other cells in the liver did not stain. No hepatocyte autoantibodies were detected, and there was no elevation of intrahepatic cytokine levels compared to uninfected controls. There were no mutations in the virus over time. A vigorous neutralizing antibody response to CVB5, Mitchell was generated, but splenic natural killer (NK) function and numbers of splenic NK cells were significantly decreased. Virus culture was positive at 3 months, but negative at further time points. Cultures were negative at 3 months for the other tissues tested. Thus, CVB5, Mitchell causes a chronic hepatitis in rabbits, with virus limited to hepatocyte cytoplasm and no evidence of autoimmunity.

The use of polymerase chain reaction (PCR) for the detection of cytomegalovirus (CMV) in bronchoalveolar lavages (BAL) of immunocompromised patients was evaluated. Firstly, PCR was compared to conventional cell culture (CCC) and shell vial assay (SVA) in 65 BAL received in our laboratory on a routine basis. Sensitivity and specificity of the PCR were 100% and 87.2% respectively. The PCR method was then applied to the monitoring of 55 BAL from nine lung transplant recipients. The data illustrate the excellent sensitivity of PCR. However the predictive value of the test for the occurrence of a symptomatic infection was weak.

Resume La place des virus dans l'etiologie des rhinopharyngites et otites aigues chez l'enfant de 0 a moins de 5 ans a ete evaluee au cours d'une enquete prospective effectuee sur 2 sites (Bordeaux et Lyon) dans l'hiver 1995–96. Les enfants vus en medecine de ville ou en consultation hospitaliere ont subi un prelevement nasopharynge achemine au laboratoire dans un milieu de transport et examine par des tests immunologiques et par mise en culture rapide. Aux 116 prelevements obtenus dans cette enquete (ORPHE), essentiellement dans les consultations hospitalieres, nous avons pu ajouter des prelevements effectues en medecine de ville dans le cadre des reseaux de surveillance de la grippe (GROGS). Globalement, 34,4 % des 285 patients atteints de rhinopharyngite febrile avaient une infection virale (Influenza A, B, VRS, Parainfluenza, ADV, Rhinovirus, Echovirus, HSV) et 29,1 % des 141 patients presentaient une otite ou une otalgie. Les otites a virus Influenza A et a VRS ont naturellement coincide avec les epidemies. Ces resultats sont en accord globalement avec les quelques donnees de la litterature. Les differences d'incidence observees tiennent : au recrutement des enfants, a leur âge, aux variations epidemiologiques des virus et a la performance des tests virologiques. Leur qualite pourrait etre amelioree par l'usage des techniques d'amplification genique. La mise en evidence de virus dans le liquide d'oreille moyenne, les infections experimentales animales, et chez l'homme adulte, confortent les hypotheses pathogeniques : le virus seul peut causer une OMA, frequemment il y a association virus + bacterie pathogene (sans qu'il y ait forcement suppuration), et l'infection virale est a l'origine de l'infection bacterienne par son action sur les cils des cellules, et en favorisant la colonisation bacterienne. La prevention par vaccination de l'infection virale chez l'enfant (grippe) a reduit significativement l'incidence de l'OMA en periode d'epidemie de grippe. Mycoplasma pneumoniae est associe a des rhinopharyngites et a des otites dans 3,5 % des cas et il est frequemment detecte en association avec des virus. Chlamydia pneumoniae n'a ete detecte que 3 fois.

The current study compares the sensitivity of RNA extraction using magnetic beads versus that of a standard extraction method. Streptavadin-coated magnetic beads were labelled with a biotinylated, enterovirus-specific oligonucleotide. RNA was extracted using labelled beads or guanidium thiocyanate-phenol-chloroform from 1, 0.1 and 0.01 TCID50/100 microliters of stock coxsackievirus types A9 and B3, echovirus type 11, enterovirus type 70 and poliovirus type 1. Each strain was tested three times. RNA extraction using magnetic beads was50% faster than the standard method. The RNA was amplified using RT-PCR, and the products were detected using agarose gel electrophoresis; 6/15 and 7/15 samples at an initial concentration of 0.01 TCID50/100 microliters were detected using magnetic beads or standard extraction, respectively. Negative-stain electron microscopy was used to determine that 0.01 TCID50/100 microliters of coxsackievirus B3 contained approximately 3 genomes. Thus, use of magnetic beads labelled with an enterovirus-specific oligonucleotide was less toxic, more rapid and as sensitive as the current standard RNA extraction method.

Thirteen laboratories participated in blind tests of a panel of 20 coded cerebrospinal fluid specimens (7 uninfected samples, 3 samples infected with 1 50% tissue culture infective dose [TCID50]/0.1 ml [nonenterovirus strains], and 10 samples infected with 10, 1, or 0.1 TCID50/0.1 ml [three different enterovirus serotypes]) on the Amplicor enterovirus PCR assay (Roche Diagnostic Systems). The panel was also evaluated by in-house PCR (two nested-PCR and three one-step PCR assay) or tissue culture (eight laboratories). The viral load was shown to influence greatly the sensitivity of the assay. The average sensitivity of the Amplicor test ranged from 67 to 98% for viral titers of 1 to 10 TCID50/0.1 ml, respectively; titers of 0.1 TCID50/0.1 ml resulted in a sensitivity of only 16%. The overall specificity of the Amplicor test was 98%. The Amplicor assay compared favorably to the five in-house PCR tests (no significant difference in either sensitivity or specificity) and was much more sensitive than tissue culture (P < 0.001), even for high viral loads. It was easy to perform, rapid (about 6 h), well-standardized, and appeared to be suitable for the diagnosis of enterovirus meningitis on a routine basis in laboratories trained in molecular biology techniques.

A new portable apparatus is described which is usable in the field to concentrate large volumes of environmental water for laboratory purposes. The portable virus concentrator consists of four parts: a sampling unit including a programmable peristaltic pump; a 5 μm prefiltration unit; an ultrafiltration system; and a physicochemical measure area. The ultrafiltration system includes 105 NMWL polysulfonate membranes, is performed at the natural pH of water and is followed by an elution step. By this technique, more than 60 l are concentrated in an hour and the recovery of three types of concentrates is observed: parasites are retained in the prefiltrate; bacteria and 80% of viruses are recovered in the concentrates.

Resume La France est un des pays ou la poliomyelite paralytique a ete eliminee. Cependant, la circulation du virus Polio n'a pas encore ete interrompue. Il est donc important de connaitre le statut immunitaire des Francais vis-a-vis de la poliomyelite. Il existe peu de donnees permettant d'evaluer le statut immunitaire de la population en France vis-a-vis des poliovirus. C'est la raison pour laquelle une enquete seroepidemiologique des anticorps neutralisants vis-a-vis des poliovirus a ete realisee chez des assures sociaux domicilies dans six departements du Centre-Ouest ayant recu un examen de sante entre janvier et avril 1991 : 300 personnes âgees de 6 a 80 ans (150 hommes, 150 femmes) ont ete tirees au sort en fonction de 6 tranches d'âge, afin d'obtenir un effectif de 50 sujets par tranche. Les anticorps neutralisants etaient recherches vis-a-vis des souches sauvages et vaccinales de reference pour les 3 types de poliovirus, ainsi que vis-a-vis de trois souches sauvages variantes. Dans la population etudiee, une proportion elevee de sujets âges de plus de 50 ans (12 a 44 %) etaient non immuns vis-a-vis de souches de type 2 ou de type 3. En particulier, 44 % des hommes de plus de 60 ans n'avaient pas d'immunite systemique vaccinale vis-a-vis du poliovirus vaccinal de type 3. Ces resultats sont en faveur de lacunes et de disparites dans l'immunite antipoliomyelitique en France. Il y a perte de l'immunite avec l'âge, non reconstituee par des rappels avec des souches vaccinales ou sauvages. Par ailleurs, les souches variantes qui continuent a circuler apparaissent encore moins bien neutralisees que les souches sauvages prototypes et les souches vaccinales. Cette situation epidemiologique et ses consequences d'ordre individuel et collectif imposent le renforcement du schema de vaccination a l'echelon de la population generale adulte et le developpement d'un systeme de surveillance performant associant la notification systematique et l'investigation des cas de paralysie flasque aigue et la recherche rationalisee des poliovirus dans l'environnement.

Resume Evaluer le statut immunologique des voyageurs avant leur depart, vis-a-vis de six maladies pouvant etre prevenues par une vaccination : tetanos, diphterie, poliomyelite, rougeole, hepatite A, hepatite B, afin de recommander les vaccinations les plus utiles. A l'occasion de leur passage dans 8 centres de vaccination anti-amarile francais, les participants ont redige un questionnaire et apres consentement ecrit, un echantillon de sang a ete preleve pour etude serologique. Entre juin et septembre 1992, 1263 sujets ont participe a l'etude, parmi lesquels 790 ont accepte de subir un prelevement sanguin. Les pourcentages des sujets immunises ont ete les suivants : rougeole : 699 (99,9 %); poliomyelite type 1 : 764 (96,7 %), type 2 : 756 (95,7 %), type 3 : 738 (93,4 %); tetanos : 764 (86,8 %); diphterie : (68 %); hepatite A : 420 (53,9 %); hepatite B : 93 (11,8 %). Chez le voyageur adulte francais les vaccinations contre l'hepatite B et la diphterie sont a recommander systematiquement s'ils se rendent en zone d'endemie. Pour l'hepatite A, un controle serologique (IgG) avant la vaccination est une alternative a la vaccination systematique chez le voyageur apres l'âge de 40 ans surtout s'il existe chez lui une notion de voyages anterieurs. Le taux de protection relativement faible contre le tetanos justifie un rappel antitetanique surtout chez les femmes, et/ou apres 40 ans. La vaccination anti-poliomyelitique dependra de l'anciennete du dernier rappel.

SUMMARYAn epidemiological study of hepatitis A and enteroviruses was conducted in a military diving training school, by evaluating the viral contamination of water using an ultrafiltration concentration technique, and assessing seroconversion and the presence of virus in stool specimens obtained from 109 divers and 48 controls. Three of 29 water specimens were positive for enterovirus by cell culture and 9 by molecular hybridization. There was little or no risk of virus infection during the training course (49 h exposure) because there was no significant difference between divers and controls for both viral isolation and seroconversion. However, a higher percentage of coxsackievirus B4 and B5 seropositive divers suggests that these were more exposed during previous water training. No hepatitis A virus (HAV) detection and no seroconversion to HAV was observed. The rate of HAV seropositive subjects was 17% in this 24·5-year-old population

Viral investigations were performed during 4 winter seasons (88/89, 89/90, 92/93, 93/94) in children attending day-care centers (DCCs) in the Rhone Département in eastern France. Over the total observation period of 4 winter seasons, 780 children were screened with a nasal swab for the presence of viruses. Of those, 230 (29.5%) had a positive viral culture. The viruses identified were respiratory syncytial virus (RSV), influenza A and B virus, parain-fluenza virus, coronavirus, rhinovirus, adenovirus and enterovirus. During that time, 83 epidemic events in 47 DCC were recorded. A particular virus was judged to be causally related to an epidemic if the identical virus was isolated in ≥ 3 children during the same outbreak of respiratory diseases. Thus, in 51 cases (61.4%) of all epidemics, the following viruses were responsible for an epidemic: RSV (n = 23), coronavirus (n = 10) (only during the season of 1993-1994), influenza A virus (n = 6), rhinovirus (n = 4), enterovirus (n = 4), adenovirus (n = 3) and parainfluenza virus (n = 1). Except for the somewhat surprising accumulation of coronavirus epidemics during the winter of 1993-1994, there were only minor seasonal variations from one year to another. As expected, RSV accounted for about one third of all respiratory tract infections in children attending DCCs and was therefore the most important single causative agent. These results are compared with data from children who did not attend a DCC and were cared for in a private practice. During the winter of 1989-1990, the viral epidemiological survey was performed at the same time and in parallel to a double-blind, placebo-controlled clinical study investigating the efficacy of OM-85 BV, an immunoactive bacterial extract. This study, enrolling 423 children attending DCCs demonstrated a protective effect of OM-85 BV in significantly reducing the risk of recurrent infections of the upper respiratory tract during the treatment period with the compound. 34% of all participating children (75 in the verum group, 70 in the placebo group) were enrolled in an additional virological study. In these patients, RSV was isolated 10 times in the placebo group, but only 5 times in the treated group (p < 0.05) and influenza A virus was present in 4 children in the placebo group, but only in 1 infant in the verum group giving a total of 14 positive virologie results in the placebo group versus 6 in the verum group (p < 0.05). Despite the small numbers of children investigated for their virologie status during respiratory infectious outbreaks, there was a statistically significant difference in the prevalence of virus carriers in favor of the children treated with OM-85 BV. These results corroborate the clinical findings.

The efficiency of seeded poliovirus and echovirus recovery from 20 litres of tap water was evaluated using a molecular ultrafiltration system. The experiments were performed using a 5μm polypropylene prefilter and two polysulfonate membranes of 105 nominal molecular weight limit. No loss of virus was observed after prefiltration. More than 96% of poliovirus were recovered after elution at pH 10.5 and 83% of echovirus after elution at pH 8.5.

The emergence of cross-resistance to various antiviral drugs was investigated both in vivo and in vitro for herpes simplex virus type 1 (HSV1) resistant to idoxuridine (IUdR 0.24%) obtained by seven successive passages (from P0 to P7) in rabbit keratitis treated by IUdR. The viral population obtained at the seventh IUdR passage (P7) showed an activity of the thymidine kinase (TK) reduced to 5.6% of the parental strain (PO); moreover, most of the clones of P7 showed an altered TK phenotype determined by the [125I]iododeoxycytidine (IDC) procedure. In rabbit keratitis, IUdR-resistant viral population P7 showed cross-resistance to bromovinyl desoxyuridine (BVDU) (0.5%) and to acyclovir (ACV) (3%). Under trifluorothymidine (1%) treatment, P7 showed an intermediate sensitivity. HSV1 at P7 remained sensitive to adenine arabinoside (Ara A) (3%) and to dihydroxy-propoxymethylguanine used at high concentration (3%). The in vitro sensitivity determination to various antiviral drugs was investigated by dye-uptake assay for the initial viral population PO and for HSV1 collected under IUdR treatment at the third (P3) and the seventh (P7) passages. Cross-resistance to TK-dependent drugs, such as IDC, BVDU, and ACV were found at P7. P7 remained sensitive to Ara A and to phosphonoformic acids antiviral drugs known not to be dependent on viral TK.

Ten sequential post-treatment herpes simplex virus type 1 (HSV-1) isolates were obtained from an immunocompromised patient whose infection, during prolonged treatment, became unresponsive to acyclovir (ACV). Of the ten isolates, eight later isolates were resistant in vitro to ACV and ganciclovir (DHPG), but remained sensitive to 9-β-D-arabinofuranosyladenine (ara-A) and phosphonoformate (PFA). Biochemical characterization of plaque-purified clones of the resistant isolates revealed an altered thymidine kinase (TK) substrate specificity phenotype. The comparative nucleotide sequence analysis of polymerase chain reaction (PCR)-amplified DNA encoding the TK genes of one sensitive and two resistant clones showed a single mutation at nucleotide 527. This change would result in a substitution of arginine by glutamine at residue 176 of the polypeptide, a mutation previously observed in a laboratory isolated variant, SC16 Tr7 (Darby et al., 1986).

The acquisition of drug resistance in vivo was investigated by 7 serial passages (from P0 to P7) of herpes simplex virus (HSV-1) in rabbit cornea treated with either IUdR (idoxuridine), IDC (idoxycytidine), ACV (acyclovir), TFT (trifluridine), or Ara A (adenine arabinoside). Therapeutic failure was acquired gradually: at P3 for IUdR, at P4 for ACV and at P5 for TFT. At P7, viral thymidine kinase (TK) activity was reduced to 5.6% of the parental strain for IUdR, to 7.5% for ACV and to 4.6% for TFT treatment. No signs of clinical unresponsiveness occurred with IDC or Ara A. The in vitro determination of antiviral drug sensitivity performed by the dye-uptake assay on HSV isolates at each passage showed a correlation between the increase in the 50% effective dose (ED50) and the increase of ulcer area grade at each passage under antiviral drug (p less than 0.1). Both IUdR- and TFT-resistant HSV1 developed cross-resistances to TK dependent drugs. However ACV-resistant HSV1 did not show cross-resistance to other antiviral TK dependent drugs. The acquisition of the cross-resistances is discussed, and the practical implications in case of therapeutic failures are suggested.

The Madin Darby Canine Kidney (MDCK) cell line is an ideal model for studying questions pertaining the influence of the environment on the epithelial phenotype biogenesis. Wild-type MDCK cells, are heterogeneous in terms of morphology and functions and consist of two genetically different subpopulations : Principal (P) cells and Intercalated (IC) cells, whose respective amounts can vary as a function-of culture conditions (culture medium, substrate, passages number...). Our previous experiments showing that MDCK cells can be easily cultivated over more than 150 passages under serum-free conditions , (Kessler et al, 1999) prompted us to follow up the genotypic and phenotypic characteristics of cells as well as the biogenesis of the epithelial phenotype during long term cultivation in such an environment.

The relative importance of the host immune response to various antigenic and functional sites on the HN glycoprotein of Sendai (6/94) virus for protection in vivo, was evaluated in mice passively immunized with monoclonal antibodies to HN and then intranasally challenged with infectious virus. Five neutralizing monoclonal antibodies reacting with distinct antigenic sites and exhibiting different reactivity patterns were selected. All of them were able to prevent entirely the growth of virus in the lungs of experimental animals injected with appropriate dilutions of monoclonal antibody. The calculation of correlation coefficients between the reduction of virus in the lungs of immunized mice and the amount of antibody, expressed in terms of hemagglutination inhibition, hemolysis inhibition or neutralizing units, showed a high degree of correlation (r = 0.89) with neutralization and a lack of correlation (r = 0.44) with hemagglutination inhibition. In parallel a minimum threshold value for protection equivalent to 2 x 10(3) neutralizing units per mouse was determined independently of the mechanism(s) by which monoclonal antibodies mediated the neutralization of the infectivity. On the HN glycoprotein of Sendai (6/94) virus we could not individualize a critical site for successful immune recognition by antibodies although the characteristics of an "ideal protective monoclonal antibody" have also been defined.

A simplified and rapid micromethod based on restriction endonuclease analysis of radiolabelled varicella-zoster virus (VZV) DNA is described and applied to strains for comparison. This procedure is cheaper and less time consuming than that requiring viral nucleocapsid purification (macromethod). The micromethod is suitable for routine DNA analysis of VZV isolates, allows differentiation of vaccine strain from wild strains, and provides evidence for variability of wild strains.

Enteroviruses were specifically detected in crude clinical specimens or in cell cultures in which the viruses were amplified by dot hybridization by using poliovirus type 1-derived, subgenomic radiolabeled cRNA probes (riboprobes). The sensitivity of this test varied from 2.5 to 33%, when clinical specimens without cell culture were examined, and was about 85% in cell culture lysates. The specificity of the test was 90 to 100%. The riboprobe corresponding to the 5'-noncoding sequence specifically detected the majority of enteroviruses (56 of 57 tested); the riboprobe derived from the VPI capsid region hybridized with the three poliovirus serotypes and with some coxsackieviruses type A and with echovirus type 7. Echovirus 22 did not hybridize with any riboprobe. In stool specimens, nasal aspirates, and cerebrospinal fluids from patients with meningitis, only one type of virus was identified in different clinical samples from the same patient by the seroneutralization test. Hybridization allowed the detection of enteroviral RNAs easily in stool specimens and nasal aspirates but with a low efficiency in cerebrospinal fluids without amplification of the viruses in cell cultures.

Adaptation of MDCK cells to serum-free conditions was performed using UltraMDCK medium (BioWHITTAKER).

Anti-neuraminidase (NA) antibodies (Ab) play a role in protection against influenza and in combination with anti-HA Ab they increase the protection in mice. To control the NA content of vaccines, which should improve vaccine standardisation and may benefit vaccine efficacy, a series of questions must be addressed: 1) The antigenic characterization of NA in vaccine strains and seed lots is based on the measurement of the enzymatic (E) activity using fetuin as substrate. The antigenic profile is established by inhibiting the E activity with post infectious ferret antisera. Overnight incubation ensures sensitivity, and fetuin substrate gives specificity by detection of variant specific antibodies. Several difficulties have to be overcome, such as the low level of E activity in MDCK grown viruses, and the lability of N1. 2) The NA protein content of the vaccines (in bulk or final product) can be measured by an ELISA capture test but the lability of the NA proteins at 4 degrees C must be checked. 3) The anti NA Ab response can be measured using a neuraminidase inhibition test. --The steric hindrance by HI antibodies does not exceed a titre of 20 in human sera. --Triton treatment of viruses reduces the steric hindrance in polyclonal sera and monoclonal antibodies but unmasks epitopes. 4) The correlations between neuraminidase inhibition, neutralization and protection, has been established in the mouse model, but remains to be shown in humans. 5) The use of a small fluorescent (MUN) or chemiluminescent (NA-STAR) substrate can be used for the rapid differentiation of N1 from N2 and NB, but not for the titration of protective NI antibodies.

Three subtypes of influenza A viruses, H1N1, H1N2 and H3N2, co-evolve in pigs in Europe. H1N2 viruses isolated from pigs in France and Italy since 1997 were closely related to the H1N2 viruses which emerged in the UK in 1994. In particular, the close relationship of the neuraminidases (NAs) of these viruses to the NA of a previous UK H3N2 swine virus indicated that they had not acquired the NA from H3N2 swine viruses circulating in continental Europe. Moreover, antigenic and genetic heterogeneity among the H1N2 viruses appeared to be due in part to multiple introductions of viruses from the UK. On the other hand, comparisons of internal gene sequences indicated genetic exchange between the H1N2 viruses and co-circulating H1N1 and/or H3N2 subtypes. Most genes of the earlier (1997–1998) H1N2 isolates were more closely related to those of a contemporary French H1N1 isolate, whereas the genes of later (1999–2000) isolates, including the HAs of some H1N2 viruses, were closely related to those of a distinct H1N1 antigenic variant which emerged in France in 1999. In contrast, an H3N2 virus isolated in France in 1999 was closely related antigenically and genetically to contemporary human A/Sydney/5/97-like viruses. These studies reveal interesting parallels between genetic and antigenic drift of H1N1 viruses in pig and human populations, and provide further examples of the contribution of genetic reassortment to the antigenic and genetic diversity of swine influenza viruses and the importance of the complement of internal genes in the evolution of epizootic strains.

Summary. "Sapporo-like viruses" (SLVs) and "Norwalk-like viruses" (NLVs) are an important cause of acute gastroenteritis in humans. While NLVs have been genetically classified into three major genetic groups consisting of 17 genetic subgroups, a classification of SLVs into comparable genetic groups remains to be determined. In an attempt to classify both SLVs and NLVs uniformly, the sequences of 2 SLV strains newly detected from French infants were analysed together with the published sequences of 9 SLV and 19 NLV strains. Distance and phylogenetic analyses were conducted on the sequences of the capsid gene, RNA polymerase gene, 3' open reading frame (3'ORF), ORF overlapping the capsid gene, and 3' untranslated region (3'UTR). The histogram showing frequency distribution of pairwise distances and the topology of the phylogenetic tree demonstrated that SLVs and NLVs could be classified uniformly on the basis of the entire capsid sequences and that the 11 SLV strains could be genetically classified into 3 major genetic groups, genogroups I, II and III, comprised of 5 genetic subgroups. The differentiation of the 11 SLV strains into these genetic groups was also maintained in the 4 remaining genome regions, while the sequences at the junction between the RNA polymerase and capsid genes were shown to be genogroup-specific.

Objective: To investigate the possible association of persistent enterovirus (EV) infection with the development of ALS. Background: Although ALS is a clinically well-defined motor neuron disease, little is known about the etiology and pathogenesis of the sporadic cases. Among the different causes that have been hypothesized, conflicting results have been reported about the possible role of persistent enteroviral infection. Methods: Reverse transcriptase-PCR (RT-PCR) and direct RT in situ PCR (RT-IS-PCR) were performed in formaldehyde-fixed spinal cord samples of 17 patients with confirmed ALS and 29 control subjects with no history of motor neuron disease. When obtained, PCR products were sequenced subsequently. Results: Using direct RT-IS-PCR, EV nucleic acid sequences were detected in 15 (88.3%) of 17 patients with ALS compared to 1 (3.4%) of 29 control subjects. PCR products were located in neuronal cell bodies of the anterior horns of the spinal cord. The RT-PCR products obtained in 13 of the 17 patients with ALS showed between 94% and 86% homology with echovirus 7 sequences. Conclusion: The 88.3% rate of detection of enterovirus (EV) nucleic acids in the neuronal cell bodies within the gray matter of the spinal cord of patients with ALS strongly suggests association between persistent EV RNA and ALS. Further work is required to confirm that the persisting EV sequences we detected are somehow involved in the development of ALS.

To achieve better standardization of influenza vaccines, an ELISA immunocapture assay was developed for N2 neuraminidase quantification. This sensitive and highly specific assay was successfully applied to vaccine preparations produced in embryonated hens' eggs from 1992 to 1997 and to antigenically related viral suspensions produced in MDCK cells. A study of the neuraminidase activity of prototype A/H3N2 strains stored at 4 degrees C showed the gradual development of enzymatic instability from 1994 onwards, accompanied by antigenic modifications of the antigen. As the phenomenon was also more pronounced with the recombinant, the question arose of the standard of immunity provided when such viruses are used for vaccination. The antibodies inhibiting neuraminidase activity in vaccinated subjects were monitored in parallel using both complete virus and purified N2 NA. The study revealed the existence of an interference phenomenon which resulted in the titre of the N1 antibodies being overestimated. The interference was due to anti-HA antibodies impeding access to the substrate at the enzymatic site by steric hindrance.

MDCK cells have been adapted to grow in a serum-free environment using Ultra-MDCK medium (BioWHITTAKER). The growth of adapted U-MDCK cells was maintained for over a year without any reduction in growth rate or modification of cell karyotype; cells were scaled up to spinner culture using several microcarriers. The cells were shown to be a very good host for influenza A and B virus replication in both the presence and absence of trypsin in the infection medium. Trypsin-independent viruses replicated to high titres (10(7)-10(8) TCID50/ml) in U-MDCK cells, after selection through serial passages without trypsin. This virus progeny exhibited uncleaved and antigenically modified haemagglutinin compared with standard viruses grown with trypsin. Finally, large amounts of influenza A and B viruses were produced in U-MDCK cells grown on microcarriers under rod-stirred conditions using selected trypsin-independent variants.