查看更多>>摘要：? 2021The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.
查看更多>>摘要：? 2021 Elsevier B.V.Reovirus fusion-associated small transmembrane (FAST) proteins induce syncytium formation. Recently, several studies have shown that the use of recombinant vectors engineered to express fusion proteins is becoming attractive for the development of enhanced oncolytic viruses. In this study, we investigated the cytotoxic effect of four different FAST proteins (p10 FAST of Avian reovirus [ARV], p10 FAST of Pulau virus [PuV], p13 FAST of Broome virus [BroV], and p14 FAST of reptilian reovirus [RRV]). Plasmids encoding FASTs were transfected into Vero cells. All FAST proteins induced syncytium formation at varying intensities. To achieve high levels of FAST expression, four different FAST genes were inserted into the murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vector. Two days after transfection in 293 T cells, only the MoMLV-10A1-p10(PuV) RCR vector showed syncytia formation. Based on these results, p10(Puv) was selected from the four FASTs. Next, we investigated the cytotoxicity of p10(PuV) on HeLa cervical carcinoma cells, HT1080 human fibrosarcoma cells, and U87 human glioma cells. Although three human cancer cell lines induced syncytium formation, U87 cells were highly susceptible to syncytia formation by transfection with p10(PuV). In addition, the viral supernatants from MoMLV-10A-p10(PuV) RCR vector-transfected 293 T cells also induced syncytium formation in HT1080, TE671, and U87 cells. This RCR vector encoding p10(PuV) is a promising candidate for cancer gene therapy.
查看更多>>摘要：? 2021 Elsevier B.V.Recently, the widespread occurrence of Sri Lankan cassava mosaic virus (SLCMV), genus Begomovirus, family Geminiviridae, which causes a mosaic disease in cassava (Manihot esculenta Crantz) in South-East Asia have, become a serious economic issue. Since cassava is propagated through vegetative cuttings, a rapid virus diagnostic method is crucial for generating virus-free planting materials. In this study, a loop-mediated isothermal amplification (LAMP) assay using six primers was developed and validated for the rapid detection of SLCMV in cassava leaves. This SLCMV assay had a detection sensitivity that was up to 10,000 times higher than that of the conventional polymerase chain reaction assay and can detect the virus from symptomless stem cutting, which is a potential long-distance spreader of the virus. Furthermore, a practical LAMP protocol using stable dried reagents from a commercial kit was established so that the assay could be performed in the field by incubating the reactions in water at 60–65 °C instead of using a thermal cycler. The primer sequences and the LAMP protocol described here should be useful for the rapid and sensitive on-site detection of SLCMV.
查看更多>>摘要：? 2021 Elsevier B.V.Bovine respiratory disease complex (BRDC) is a serious disease affecting feedlot cattle in China and likely other places worldwide. Bovine viral diarrhea virus (BVDV) and bovine parainfluenza virus type 3 (BPIV3) are principally responsible for causing BRDC, and are a major strain to the industrial economy. Eradication of these viruses/disease requires swift viral identification and treatment. Hence, this study established a fast and easy procedure of BVDV and BPIV3 identification that employs reverse transcription recombinase polymerase amplification (RT-RPA) and lateral flow dipstick (LFD), and uses primers and lateral flow (LF) probe targeting the 5′-UTR gene of BVDV and phosphoprotein P gene of BPIV3, respectively. Our assay was able to successfully amplify BVDV and BPIV3 RNA within 25 min at 35 °C using RT-RPA, with products visible on the LFD within 5 min at room temperature (RT). The lowest detection limits were 50 RNA molecules for BVDV and 34 RNA molecules for BPIV3 per reaction. We also demonstrated that the established dual RT-RPA LFD assay was precise and targeted, harboring excellent potential to become an onsite molecular diagnostic tool in the detection of BVDV and BPIV3. This method can detect BVDV (Pestivirus A, B) and BPIV3, and exhibit no cross-reaction with other viruses like the classical swine fever virus (CSFV) and infectious bovine rhinotracheitis virus (IBRV). The assay performance was further assessed with clinical samples, and demonstrated good performance in comparison to real-time RT-PCR (RT-qPCR). Moreover, the RT-RPA LFD assay was comparitively rapid and required minimal training.
查看更多>>摘要：? 2021 Elsevier B.V.The hepatoma cell lines stably expressing sodium taurocholate cotransporting polypeptide (NTCP), the receptor of hepatitis B virus (HBV) infection, serve as important infection models for studying viral biology and drug discovery. However, the efficiency of infection greatly varies. In this study, we studied the effects and potential mechanisms of Matrigel? hESC-qualified (M-hq), a biological basement membrane matrix commonly used in cell culture, on promotion HBV in vitro infection in HepG2-NTCP cells. For the first time, our findings demonstrate that M-hq could enhance the infection efficiency of cell culture-derived HBV with no impact on the cell viability, the HBV transcription and response to antiviral treatments. The infection enhancement is reproducible and is suggested to occur at HBV attachment step. Our study suggests that this novel system is applicable for studying HBV biology and new drugs.
查看更多>>摘要：? 2021 The AuthorsWaterborne epidemics of human hepatitis virus A and E (HAV and HEV) have been reported worldwide. Molecular biology techniques, such as reverse transcription polymerase chain reaction (RT-PCR), have been widely used to detect the two hepatitis viruses. However, comparative studies of various types of samples are needed, and different environmental factors, including the low copy pathogens, presence of PCR inhibitors in the sample, unknown non-specific reaction with template, and sequence diversity leading to new variants in viruses, should be considered. In addition, standard positive material is required to determine the accuracy of the PCR and should be able to distinguish between false and real positives. In this study, we developed RT-PCR primer sets and optimised standard templates for HAV and HEV detection to address the above concerns associated with test sensitivity and possible PCR inhibition. Finally, previously reported diagnostic methods of HAV and HEV were compared and an applicability test using groundwater was performed. The nested RT-PCR developed in this study is expected to contribute to assess water safety by monitoring HAV and HEV in non-disinfected water, like groundwater.
查看更多>>摘要：? 2021Bovine viral diarrhea virus (BVDV) comprises two species, BVDV-1 and BVDV-2. But given the genetic diversity among pestiviruses, at least 22 subgenotypes are described for BVDV-1 and 3-4 for BVDV-2. Genetic characterization is generally accomplished through complete or partial sequencing and phylogeny, but it is not a reliable method to define antigenic relationships. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to define antigenic relatedness can be difficult to discern for BVDV isolates within the same BVDV species. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between US vaccine strains and field isolates from Switzerland, Italy, Brazil, and the UK. Polyclonal sera were generated against six BVDV strains currently contained in vaccine formulations, and each serum was used in VNs to measure the titers against seven vaccine strains (including the six homologous strains) and 23 BVDV field isolates. Principal component analysis (PCA) was performed using VN titers, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among various isolates suggesting antigenic relatedness. As expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. Notably, a number of clusters representing antigenically related BVDV-1 subgroups contain isolates of different subgenotypes. The multivariate analysis may be a method to better characterize antigenic relationships among BVDV isolates that belong to the same BVDV species and do not have distinct antigenic differences. This might be an invaluable tool to ameliorate the composition of current vaccines, which might well be important for the success of any BVDV control program that includes vaccination in its scheme.
查看更多>>摘要：? 2021 Elsevier B.V.In Emergency Room, Point-of-care antigen testing for SARS-CoV-2 antigen can expedite clinical strategies for patient management. We tested 1,232 consecutive patients during Italian second wave peak using the recent LumiraDx microfluidic assay. This assay showed high concordance (96.9 %), sensitivity and specificity compared to molecular testing, being highly valuable.
查看更多>>摘要：? 2021Molecular identification and characterization of novel or re-emerging infectious pathogens is critical for disease surveillance and outbreak investigations. Next generation sequencing (NGS) using Sequence-Independent, Single-Primer Amplification (SISPA) is being used extensively in sequencing of viral genomes but it requires an expensive library preparation step. We developed a simple, low-cost method that enriches nucleic acids followed by a ligation-free (LF) 2-step Polymerase Chain Reaction (PCR) procedure for library preparation. A pan-chimeric universal primer (JS15N14) containing 15 nucleotides with a random tetradecamer (14N) attached to the 3′-end was designed. The complimentary primer (JS15) was used for nucleic acid enrichment in a first round PCR. A second PCR was designed to create Illumina sequencer-compatible sequencing-ready libraries for NGS. The new LF-SISPA protocol was tested using six RNA and DNA viral genomes (10.8–229.4 kilobases, kb) from an ATCC virome nucleic acid mix (ATCC? MSA-1008?) followed by analysis using One Codex, an online identification tool. In addition, a human stool sample known to be positive for norovirus GII was sequenced, and de novo assembly was performed using the Genome Detective Virus Tool allowing for near complete genome identification in less than 24 h. The LF-SISPA method does not require prior knowledge of target sequences and does not require an expensive enzymatic library preparation kit, thereby providing a simple, fast, low-cost alternative for the identification of unknown viral pathogens.
查看更多>>摘要：? 2021Adenoviruses (AdVs) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney 239 (HEK293) or PER.C6 virus producer cells with AdV plasmid vectors or infecting these cells withcell lysates containing replication-defective AdV. Cell lysates can be purified further by caesium chloride or chromatographic protocols to research virus seed stocks (RVSS) for characterisation to high quality master virus seed stocks (MVSS) and working virus seed stocks (WVSS) before downstream production of pure, high titre AdV. Lysates are poorly infectious, block filtration columns and have limited storage capability. Aqueous two-phase systems (ATPS) are an alternative method for AdV purification that rapidly generates cleaner RVSS for characterisation to MVSS. After testing multiple ATPS formulations, an aqueous mixture of 20 % PEG 600 and 20 % (NH4)2SO4 (w/w) was found most effective for AdV partitioning, producing up to 97+3% yield of high-titre virus that was devoid of aggregates both effective in vitro and in vivo with no observable cytotoxicity. Importantly, AdV preparations stored at ?20 °C or 4 °C show negligible loss of titre and are suitable for downstream processing to clinical grade to support the need for AdV vaccines.
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