A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

BACKGROUND: Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition. RESULTS: A SYBR Green real-time RT-PCR assay was developed making use of a foreign internal RNA control added in the same tube. This assay is able to detect human and bovine noroviruses belonging to genogroups I, II and III and to distinguish between norovirus and internal control amplicons using melting curve analysis. A 10-fold dilution of samples appears to be the method of choice to remove inhibition. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. CONCLUSION: This SYBR Green real-time RT-PCR assay allows the detection of the most important human and bovine noroviruses in the same assay, and avoids false negative results making use of an internal control. Melting curves allow the discrimination between the internal control and norovirus amplicons. It gives preliminary information about the species of origin. The sensitivity of the developed assay is higher than conventional RT-PCR and a 10-fold dilution of samples showed a better efficiency and reproducibility to remove RT-PCR inhibition than addition of bovine serum albumin

Computer-assisted total knee arthroplasty: Comparative results in a preliminary series of 72 cases

The aim of this study was to assess the value of navigation in achieving correct positioning of the implants and soft-tissue balance in total knee arthroplasty. We compared the axis alignment achieved in 72 LCS TKA's performed with navigation assistance to a historical cohort of 62 LCS TKA's implanted with the conventional instrumentation. The position of the tibial and femoral implants and the post-operative mechanical axes of the lower limbs were compared in the two series: there was a highly significant improvement in the alignment accuracy in the navigated series (p < 0.0001). There were no outliers in the computer-assisted series whereas 47% of the cases in the conventional series showed deviations of the mechanical axis of the lower limb of more than 2° from neutral alignment. However, the position of the femoral implants in rotation was not improved, suggesting that there may be a need for a more refined technique for rotational alignment of the femur, which was based essentially in the present series on ligament balance in flexion. The height of the joint line was preserved in 89% of the cases, validating the empirical use of the spreader tool prototype used during the study. Navigation eases optimal ligament balancing, by providing information that is used for appropriate release of soft tissue to achieve the proper mechanical axis. No major complication related to the use of navigation was observed. Operation time was lengthened on average by 30 minutes. Long-term studies are necessary to show whether better accuracy in ligament balancing and higher precision in restoration of mechanical axes will improve the functional results and the survival rate of knee arthroplasty. © 2005, Acta Orthopædica Belgica.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Genetic and evolutionary perspectives on genogroup III, genotype 2 bovine noroviruses

Bovine noroviruses are enteric pathogens that are detected in stool samples from cattle. Five genogroups are currently described in the genus Norovirus (family Caliciviridae), and within the genogroups, sequences are further divided into genotypes according to genetic homology and phylogenetic relationships. In this study, stool specimens from Belgian cattle were screened by RT-PCR. All of the sequences that were detected were phylogenetically related to genogroup III genotype 2 bovine noroviruses, confirming their higher prevalence in comparison with strains from genotype 1. When other sequences from around the world were introduced, phylogenetic inferences allowed neither the determination of phylogenetic lineages over time nor the deduction of topotypes for genotype 2 bovine noroviruses. Three complete genotype 2 bovine norovirus sequences were also compared genetically (Newbury2/1976 /UK, Dumfries/1994/UK and B309/2003/BE). Interestingly, the genetic divergence of the complete genomes of these three strains was relatively low, but a region of the N-terminal protein encoded by ORF1, the hypervariable region of the capsid gene encoded by ORF2, and a region of the minor structural protein encoded by ORF3 seem to be the most exposed to genetic evolution. Bayesian inference also showed that genetic evolution of genogroup III, genotype 2 bovine noroviruses over a 30-year period seemed to be lower than that already reported for noroviruses from the genotypes 3 and 4 in genogroup II

Validity of Gerber’s subscapularis test

info:eu-repo/semantics/nonPublishe

Détection des sapovirus porcins par RT-PCR en temps réel

Les sapovirus appartiennent à la famille virale des Caliciviridae. Ces virus sont responsables de gastroentérites épidémiques dans l’espèce humaine et sont actuellement majoritairement détectés en Asie. Des souches de sapovirus ont également été isolées dans l’espèce porcine. Trois pays européens seulement ont rapporté la présence de souches de sapovirus porcins dans leurs troupeaux: la Hongrie (Reuter et al., 2007), l’Italie (Martella et al., 2008) et tout récemment la Belgique (Mauroy et al., 2008). La détection moléculaire de la présence de séquences de sapovirus porcins dans des pays où densités d’élevages et de population humaine se conjuguent posent des questions d’ordre zoonotique, problème déjà en discussion pour des virus qui leur sont proches: les norovirus humains et animaux (Scipioni et al., 2008). De plus l’identification de ces nouveaux pathogènes pour l’espèce porcine suggèrent également d’en évaluer les impacts économique, sanitaire et clinique pour cette filière. Ces questions ne pourront être correctement évaluées que si ces virus sont recherchés et que des méthodes fiables de détection sont développées. Dans une précédente étude (Mauroy et al., 2008), le couple d’amorce p289/p290, développé par Jiang et collaborateurs (1999) pour la détection des calicivirus humains (norovirus et sapovirus), avait permis la détection de séquences génomiques de sapovirus et de norovirus porcins. Les amorces p289/290 ont été utilisées dans cette étude dans une RT-PCR en temps réel mettant à profit la technologie SYBR green. L’étude des courbes de dissociation obtenues nous a permis de pouvoir différencier des échantillons de matières fécales positifs pour la présence de séquences génomiques de sapovirus porcins de ceux qui étaient positifs pour la présence de différents calicivirus humains ou animaux (norovirus humains, sapovirus humains, norovirus bovin et porcin, vésivirus félin isolé de tractus respiratoire, vésivirus félin isolé de tractus digestif). Cette méthode devra être dans un premier temps appliquée à un échantillon plus important de matières fécales confirmées positives pour la présence de sapovirus porcins pour pouvoir être validée. La validation de cette méthode pourra ensuite permettre aux laboratoires de diagnostic de disposer d’une méthode rapide et fiable de détection de ces virus dans les filières concernées

Virus-like Particles of Human and Bovine Noroviruses

1.Introduction and Objectives Noroviruses have a positive-sense, single-stranded RNA genome of about 7.5 kb containing three open reading frames (ORFs). ORF1 encodes the non-structural proteins, ORF2 encodes the single capsid protein with a molecular weight of 56 kD and ORF3 encodes a minor structural protein. These viruses are very stable and successful infection can be achieved even with very low infectious dose. Human noroviruses are the major cause of non bacterial, food-borne gastroenteritis worldwide. Contamination is usually oro-fecal (Green et al. 2001). Belonging to the Caliciviridae family, the genus Norovirus also contains viruses infecting animals, in particular bovines. This genus is divided into five genogroups (Ando et al. 2000). The existence of bovine noroviruses raises the question of their possible zoonotic character; the viruses could be transmitted to human by contaminated effluents for example (Scipioni et al., this congress). The study of norovirus is hampered by the lack of a cell culture system. However virus like particles (VLPs) can be produced and they are antigenically and morphologically similar to the wild virus (Jiang et al. 1992). The aim of this work was to obtain VLPs from human and bovine strains. Such VLPs will be used as antigens to produce specific polyclonal and monoclonal antibodies. 2.Material and Methods The capsid protein genes of a human norovirus strain (H384) and a bovine strain (B309) were amplified by RT PCR from stools collected from human and veterinary diagnostic laboratories. These sequences were analysed by the Basic Local Alignment Search Tool (BLAST) available on the NCBI website. Plasmids containing the ORF2 sequence of two reference human strains, the Norwalk (NV, belonging to the genogroup I) and the Hawaii (HV, belonging to the genogroup II) strains, were kindly received from Jan Vinje (University of North Carolina, USA). The baculovirus protein expression system (Invitrogen®) was used to obtain VLPs. Briefly, cells and supernatants were harvested after freezing/thawing cycles. Cells were pelleted. VLPs were purified from supernatants by ultracentrifugation through a 30% sucrose cushion and then by isopycnic ultracentrifugation in CsCl. Fractions resulting from the gradient were analysed by SDS-PAGE. Protein assembly into VLPs was checked by electron microscopy. 3.Results H384 strain ORF2 is closely related to the HuNV/Altenkirchen 140/01/DE strain (99% of identity). Thus, H384 most likely belongs to genogroup II genotype 4 (GII.4, the Lordsdale group). B309 capsid gene is closely related to genogroup III genotype 2 (Newbury2). Electron microscopy confirmed the presence of norovirus-like particles in the supernatant of recombinant baculovirus infected cell culture. By SDS PAGE applied on the fractions resulting of the CsCl gradient, a protein of 56 kD was detected and confirmed the expression of the capsid protein. Therefore, VLPs were obtained for all the 4 strains investigated. 4.Discussion and Conclusions Currently we dispose of representative surrogates of three norovirus genogroups (GI, GII and GIII). H384 (GII.4) is a representative of the most commonly diagnosed genotype from current outbreaks in many countries (Blanton et al. 2006). These VLPs will therefore serve as antigens to raise polyclonal and monoclonal antibodies. These tools will be used for the diagnosis of the related infections in human and domestic animals

Detection and quantification of human and bovine novoviruses by a TaqMan RT-PCR assay with a control for inhibition.

Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and calves. Reverse transcription-polymerase chain reaction (RT-PCR) has become the ‘‘gold standard’’ for detection of noroviruses in faecal and environmental samples. However, false negative results due to co-concentration of RT-PCR inhibitors are a continuous concern. A TaqMan real-time RT-PCR assay making use of a foreign internal RNA control and a RNA standard was developed. Very interestingly, this method is capable of detecting human noroviruses belonging to genogroups I and II, and bovine noroviruses belonging to genogroup III. Inhibitors were removed efficiently by 1/10 dilution of the sample or addition of bovine serum albumin to the RT-PCR mix. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. The ability to detect norovirus in stool samples that were negative by conventional RT-PCR assay demonstrate the higher sensitivity of the TaqMan assay compared to the conventional RT-PCR assay. This real-time RT-PCR assay allows the detection of both human and bovine noroviruses, avoids false negative results and is able to quantify the level of norovirus contamination.RF6/85 et SD/AF/0

Production de pseudoparticules de norovirus humains et bovins et leur utilisation diagnostique

Les norovirus sont des agents majeurs de gastroentérite humaine d’origine alimentaire et ceci à travers le monde entier. La contamination est habituellement oro-fécale. Les norovirus sont non enveloppés et ont un génome composé d’ARN monocaténaire de polarité positive d’approximativement 7,5 kb. Au sein de ce génome, trois cadres ouverts de lecture (ORFs) sont décrits. L’ORF 1 code pour une polyprotéine qui sera par la suite clivée pour donner les différentes protéines non-structurales. L’ORF 2 encode pour l’unique protéine de capside. Une protéine structurale mineure dont le rôle est encore peu caractérisé est encodée par l’ORF 3. Ces virus sont très résistants dans l’environnement et une infection peut survenir même avec une très faible dose infectieuse. Appartenant à la famille des Caliciviridae, le genre norovirus est composé de cinq génogroupes (G) et contient également des virus infectant les animaux (bovins, porcins, murins). L’impact des norovirus animaux et plus particulièrement des norovirus bovins peut être envisagé à travers un possible caractère zoonotique de ces virus ; ainsi ils pourraient par exemple être transmis à l’homme via des eaux d’effluent contaminées. L’étude des norovirus humains et bovins est encore à l’heure actuelle entravée par l’absence d’un système de culture cellulaire. Cependant différents systèmes d’expression protéique ont été utilisés afin d’exprimer la protéine de capside, celle-ci s’assemblant spontanément avec d’autres pour reformer des pseudoparticules virales ou virus-like particles (VLPs). Ces VLPs sont morphologiquement et antigéniquement semblables aux virus natifs. Le but de ce travail était dans un premier temps d’obtenir des VLPs de souches humaines et bovines. Les gènes codant pour la protéine de capside d’une souche humaine (H384) et d’une souche bovine (B309) ont été amplifiés par RT-PCR à partir d’échantillons de matières fécales collectés dans des laboratoires de diagnostic humain et vétérinaire. Ces gènes ont été séquencés et comparés grâce à des outils informatiques (Basic Local Alignment Search Tool, BLAST) à la banque de séquences déjà disponibles sur le site du NCBI. Nous avons pu déterminer que la séquence de capside de la souche H384 était proche de celle de la souche HuNV/Altenkirchen 140/01/DE, une souche de GII et de génotype 4 tandis que la séquence de la souche B309 était proche de celles de GIII et de génotpe 2 (groupe Newbury2). Des plasmides contenant les séquences des ORFs 2 de deux souches de norovirus humains de référence, la souche de Norwalk (GI) et celle de Hawaii (GII), ont été aimablement fournies par le Docteur Jan Vinjé (CDC, Atlanta, USA). Le système d’expression protéique baculovirus (Invitrogen) a été utilisé pour obtenir des VLPs. Brièvement, des cellules d’insecte des lignées Sf9 et H5 ont été infectées avec des baculovirus recombinants pour la protéine de capside des norovirus. Les VLPs ont été purifiées à partir des surnageants par ultracentrifugation sur un coussin de sucrose à 30% suivie d’une ultracentrifugation en gradient de chlorure de césium. Ce gradient a été fractionné et les différentes fractions ont été analysées pour la présence de la protéine de capside des norovirus par SDS-PAGE. Pour vérifier que les fractions positives contenaient bien des VLPs, les surnageants ont été observés en microscopie électronique. Nous avons ainsi obtenu des VLPs de norovirus de trois des cinq génogroupes décrits (GI, II et III). Ces VLPs ont été utilisées pour immuniser des souris afin de produire des anticorps monoclonaux et pour immuniser des lapins afin d’obtenir des anticorps polyclonaux. Nous avons pu aussi tester une banque de sérums bovins par ELISA pour la présence d’anticorps dirigés contre les souches de norovirus de génogroupe III et de génotype 2, les plus fréquemment mises en évidence au sein de notre banque de matières fécales bovines

Noroviruses and sapoviruses in pigs in Belgium.

Porcine noroviruses and sapoviruses belong to the family Caliciviridae and are rarely reported in European countries. In this study, swine stools from a region representative of northern Europe were screened for these viruses by RT-PCR. Both porcine noroviruses and sapoviruses were detected, showing their circulation in this region. The porcine norovirus strains were genetically related to genotype 19 strains in the genogroup II of the genus Norovirus. The porcine sapovirus strains were genetically related to the porcine enteric calicivirus Cowden reference strain and to newly described porcine strains in the genus Sapovirus