Rapid bedside inactivation of Ebola virus for safe nucleic acid tests

Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available MagNA Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding MagNA Pure lysis/binding buffer directly into vacuum blood collection EDTA-tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months and Ebola virus RNA is preserved in the MagNA Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from MagNA Pure lysis/binding buffer-inactivated samples using the QIAamp Viral RNA mini kit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection

Vector competence of British Culicoides species for Bluetongue virus serotype 8

The proportion of infected vectors which are able to transmit an arbovirus to a susceptible host has a significant impact on the epidemic potential of such a virus. Assessing vector competence is therefore crucial to evaluate accurately the risk posed by such a disease to any non-endemic region. The vector competence of various Culicoides species in Scotland for bluetongue virus serotype 8 (BTV-8) was assessed by a pad-feeding technique, and a high-throughput virus extraction and isolation procedure. This was coupled with a multiplex polymerase chain reaction (PCR) to identify members of the Culicoides Obsoletus complex to species level. These results are compared with vector competence results of further Culicoides Obsoletus in South-East England assessed by the same method. A very low level of competence for this strain was detected in all Culicoides species tested, similar to that described for this strain in C. imicola originating from both Corsica and the Onderstepoort Veterinary Institute in South Africa. The implications of this are discussed in relation to future studies and also with regard to wider aspects of orbivirus transmission in the European Union. (Texte intégral

Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

BACKGROUND Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample. RESULTS EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10² to 1.3 × 10⁸ copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³ to 2.0 × 10⁵ copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴ to 1.1 × 10⁵ copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10² to 5.6 × 10³ copies/ml in HIV-infected patients (n = 12), and 2.0 × 10² to 9.1 × 10⁴ copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11). CONCLUSION Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.The Ausimmune Study is funded by the National Multiple Sclerosis Society of the USA, the National Health & Medical Research Council (Project Grant 316901) and Multiple Sclerosis Research Australia

Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community

Abstract Recent studies have conflicting data regarding the presence of intra-amniotic microbiota. Viral communities are increasingly recognized as important although overlooked components of the human microbiota. It is unknown if the developing fetus is exposed to a community of viruses (virome). Given the debate over the existence of an intra-amniotic microbial community and the importance of understanding how the infant gut is populated, we characterized the virome and bacterial microbiota of amniotic fluid from 24 uncomplicated term pregnancies using next-generation sequencing methods. Contrary to expectations, the bacterial microbiota of amniotic fluid was indistinguishable from contamination controls. Viral reads were sparse in the amniotic fluid, and we found no evidence of a core viral community across samples

VirusPKT: A Search Tool For Assimilating Assorted Acquaintance For Viruses

Viruses utilize various means to circumvent the immune detection in the biological systems. Several mathematical models have been investigated for the description of viral dynamics in the biological system of human and various other species. One common strategy for evasion and recognition of viruses is, through acquaintance in the systems by means of search engines. In this perspective a search tool have been developed to provide a wider comprehension about the structure and other details on viruses which have been narrated in this paper. This provides an adequate knowledge in evolution and building of viruses, its functions through information extraction from various websites. Apart from this, tool aim to automate the activities associated with it in a self-maintainable, self-sustainable, proactive one which has been evaluated through analysis made and have been discussed in this paper

Characterization of a monoclonal antibody to turnip mosaic virus and its use in immunodiagnosis of infection

Des anticorps monoclonaux spécifiques au virus de la mosaïque du panais (TuMV) ont été produits et utilisés dans un bioessai en sandwich à double anticorps afin de détecter des virus dans des plantes infectées. Un anticorps particulier d'un clone hybridome ayant les caractéristiques recherchées de croissance, de spécificité et de production d'anticorps a été décrit. Cet anticorps a été montré par microscopie électronique immunocytochimique and par immunodétection en point comme réagissant avec une protéine d'enrobage d'un virion. Les conditions procurant une extraction efficace du virus à partir des feuilles ont été étudiées par l'utilisation de l'anticorps dans les étapes de capture et de détection du bioessai en sandwich. Avec un système de tampons d'extraction contenant plusieurs détergents, un essai très sensible a été produit qui détecte des virus de façon fiable dans les plantes infectées. Cet essai est maintenant utilisé de façon routinière pour l'immunodiagnostic des infections causées par le virus de la mosaïque du panais.Monoclonal antibodies specifie for turnip mosaic virus (TuMV) were produced and used in a double antibody sandwich enzyme immunoassay to detect virus in infected plants. One particular antibody from a hybridoma clone having desirable growth, specificity and antibody production properties was characterized in detail. This antibody was shown by immunocytochemical electron microscopy and immunoblotting to react with a virion coat protein. Conditions providing efficient extraction of virus from leaves were investigated by using the antibody in both capture and detection steps of a sandwich immunoassay. With an extraction buffer System containing multiple detergents, a highly sensitive assay was produced that reliably detected virus in infected plants. This assay is now in routine use for immunodiagnosis of turnip mosaic virus infections

Characterisation and detection of dasheen mosaic potyvirus in Zantedeschia : a thesis presented in partial fulfilment of the requirements for the degree of Master of Horticultural Science in Plant Health at Massey University, Palmerston North, New Zealand

Four potyvirus isolates believed to be dasheen mosaic potyvirus, the most frequently occurring virus to infect members of the Araceae, were obtained from Caladium, Colocasia, Xanthosoma and Zantedeschia in world-wide locations. Properties of these isolates such as particle length, serological relatedness, electrophoretic mobility of coat proteins and genomic characteristics were compared. Serologically distinct strains of dasheen mosaic potyvirus were apparent amongst the isolates. The difference in the serological relationship was coupled with a variation in symptom expression. An isolate from Colocasia esculenta (L.) Schott was not serologically related to the other isolates. Further isolates from C. esculenta also exhibited no relationship. The modal length was different as well as the ability of complementary deoxyribonucleic acid, produced to the viral ribonucleic acid, to bind with some of the primers used in the polymerase chain reaction. This evidence led to the proposal that the isolate from C. esculenta was not dasheen mosaic potyvirus; this virus is tentatively named taro feathery mottle potyvirus. Cytoplasmic inclusion protein aggregates of dasheen mosaic potyvirus were purified from infected leaf tissue. SDS-polyacrylamide gel electrophoretic analysis of samples revealed a major band with an estimated molecular weight of 68,000 daltons. Such a band was absent from healthy tissue samples. The ATPase activity in samples from each purification step was determined by measuring the amount of [32P] released from the [λ-32P]ATP during incubation with the cytoplasmic inclusion protein. The level of ATPase activity in each sample showed a strong correlation with the amount of protein that was present. In a limited survey of commercial plantings twenty nine tubers grown for cutflower or tuber export were obtained from seven properties at different locations in New Zealand and grown on in a greenhouse. Each plant was indexed for virus infection. Electron microscopy revealed that plants from three of the properties contained 720nm flexuous rods. Samples from all but two plants tested positive to a potyvirus group antiserum using the enzyme-linked immunosorbent assay. The remaining two plants tested positive in microprecipitin and rapid immune electron microscopy tests to an antiserum prepared to a member of the carlavirus group. Particles from these plants were mechanically transmitted to Nicotiana tabacum 'Havana'. Rod-shaped particles of 300nm were observed in plants from four properties and tested positive to tobacco mosaic tobamovirus antiserum using a microprecipitin test. While inoculations to herbaceous indicators resulted in no symptoms, 300nm particles were observed in samples from the indicator plants. Tomato spotted wilt tospovirus, potato X potexvirus and cucumber mosaic cucumovirus, reported to infect Zantedeschia spp, were not detected

Single genome sequencing of near full-length HIV-1 RNA using a limiting dilution approach

Sequencing very long stretches of the HIV-1 genome can advance studies on virus evolution and in vivo recombination but remains technically challenging. We developed an efficient procedure to sequence near full-length HIV-1 RNA using a two-amplicon approach. The whole genome was successfully amplified for 107 (88%) of 121 plasma samples including samples from patients infected with HIV-1 subtype A1, B, C, D, F1, G, H, CRF01_AE and CRF02_AG. For the 17 samples with a viral load below 1000 c/ml and the 104 samples with a viral load above 1000 c/ml, the amplification efficiency was respectively 53% and 94%. The sensitivity of the method was further evaluated using limiting dilution of RNA extracted from a plasma pool containing an equimolar mixture of three HIV-1 subtypes (B, C and CRF02_AG) and diluted before and after cDNA generation. Both RNA and cDNA dilution showed comparable sensitivity and equal accuracy in reflecting the subtype distribution of the plasma pool. One single event of in vitro recombination was detected amongst the 41 sequences obtained after cDNA dilution but no indications for in vitro recombination were found after RNA dilution. In conclusion, a two-amplicon strategy and limiting dilution of viral RNA followed by reverse transcription, nested PCR and Sanger sequencing, allows near full genome sequencing of individual HIV-1 RNA molecules. This method will be a valuable tool in the study of virus evolution and recombination

A Practical Incremental Learning Framework For Sparse Entity Extraction

This work addresses challenges arising from extracting entities from textual data, including the high cost of data annotation, model accuracy, selecting appropriate evaluation criteria, and the overall quality of annotation. We present a framework that integrates Entity Set Expansion (ESE) and Active Learning (AL) to reduce the annotation cost of sparse data and provide an online evaluation method as feedback. This incremental and interactive learning framework allows for rapid annotation and subsequent extraction of sparse data while maintaining high accuracy. We evaluate our framework on three publicly available datasets and show that it drastically reduces the cost of sparse entity annotation by an average of 85% and 45% to reach 0.9 and 1.0 F-Scores respectively. Moreover, the method exhibited robust performance across all datasets.Comment: https://www.aclweb.org/anthology/C18-1059