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Abstract Background Virus genome sequences, generated in ever-higher volumes, can provide new scientific insights and inform our responses to epidemics and outbreaks. To facilitate interpretation, such data must be organised and processed within scalable computing resources that encapsulate virology expertise. GLUE (Genes Linked by Underlying Evolution) is a data-centric bioinformatics environment for building such resources. The GLUE core data schema organises sequence data along evolutionary lines, capturing not only nucleotide data but associated items such as alignments, genotype definitions, genome annotations and motifs. Its flexible design emphasises applicability to different viruses and to diverse needs within research, clinical or public health contexts. Results HCV-GLUE is a case study GLUE resource for hepatitis C virus (HCV). It includes an interactive public web application providing sequence analysis in the form of a maximum-likelihood-based genotyping method, antiviral resistance detection and graphical sequence visualisation. HCV sequence data from GenBank is categorised and stored in a large-scale sequence alignment which is accessible via web-based queries. Whereas this web resource provides a range of basic functionality, the underlying GLUE project can also be downloaded and extended by bioinformaticians addressing more advanced questions. Conclusion GLUE can be used to rapidly develop virus sequence data resources with public health, research and clinical applications. This streamlined approach, with its focus on reuse, will help realise the full value of virus sequence data.

Background: Virus genome sequences, generated in ever-higher volumes, can provide new scientific insights and inform our responses to epidemics and outbreaks. To facilitate interpretation, such data must be organised and processed within scalable computing resources that encapsulate virology expertise. GLUE (Genes Linked by Underlying Evolution) is a data-centric bioinformatics environment for building such resources. The GLUE core data schema organises sequence data along evolutionary lines, capturing not only nucleotide data but associated items such as alignments, genotype definitions, genome annotations and motifs. Its flexible design emphasises applicability to different viruses and to diverse needs within research, clinical or public health contexts. Results: HCV-GLUE is a case study GLUE resource for hepatitis C virus (HCV). It includes an interactive public web application providing sequence analysis in the form of a maximum-likelihood-based genotyping method, antiviral resistance detection and graphical sequence visualisation. HCV sequence data from GenBank is categorised and stored in a large-scale sequence alignment which is accessible via web-based queries. Whereas this web resource provides a range of basic functionality, the underlying GLUE project can also be downloaded and extended by bioinformaticians addressing more advanced questions. Conclusion: GLUE can be used to rapidly develop virus sequence data resources with public health, research and clinical applications. This streamlined approach, with its focus on reuse, will help realise the full value of virus sequence data. [ABSTRACT FROM AUTHOR]

White spot syndrome virus ( WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007-2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats ( VNTR) located in open reading frame ( ORF) 75, 125 and 94. WSSV genotype was characterized as (X14/15, X23/24) (N75-N125-N94) where X is the number of base pair deletion in the variable region and N is the number of repeat units ( RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (595014/15, 1097123/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (595014/15, 1097123/24) (375-6125-794). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries. [ABSTRACT FROM AUTHOR]

Background Herpes Simplex Virus (HSV) drug resistance is a significant public health concern among immunocompromised individuals. Phenotypic assays are considered the gold standard method for detecting HSV drug resistance. However, plaque reduction assays (PRAs) are technically demanding, often with long turnaround times of up to four weeks. In contrast, genotypic tests can be performed within a few days. Objectives The development and coordination of the first European External Quality Assessment (EQA) study to evaluate phenotypic and genotypic methods used for HSV drug resistance testing in specialised reference laboratories. Study design Four HSV-1 or HSV-2 strains with different antiviral susceptibility profiles were isolated from clinical samples. Isolates were quantified by qPCR, and aliquoted in culture medium. One isolate was distributed at two dilutions to help assess assay sensitivity. The panel was distributed to five European centres with a six-week deadline for the return of phenotypic and genotypic results, together with clinical reports. Results Four out of five participating labs returned results by the deadline. Limited results were later available from the fifth lab. Phenotypic and genotypic data were largely, but not completely, concordant. An unusual resistance profile shown by one of the samples was explained by the detection of a mixed virus population after extensive further investigation by one of the centres. Conclusions Discordant clinical outputs reflecting the diversity of phenotypic methodologies demonstrated the utility of this exercise. With emerging genotypic technologies looking to supplant phenotyping, there is a need for curated public databases, accessible interpretation tools and standardised control materials for quality management. By establishing a network of testing laboratories, we hope that this EQA scheme will facilitate ongoing progress in this area. [ABSTRACT FROM AUTHOR]

Influenza is a contagious respiratory illness caused by influenza viruses which possess the enormous threat to older people and young children. Rapid and precise discrimination of virus subtypes are quite crucial for the early therapy, prophylaxis and the prevention of epidemic outbreaks. Herein, a universal strategy, with influenza A virus (IAV) as a model, is proposed for the discrimination of virus subtypes based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Reference library based on nine IAVs subtypes (i.e., H1N1, H3N2, H4N8, H5N8, H6N6, H7N7, H9N2, H10N8, and H11N8) was set up for matching various IAVs subtypes. The simulative test spectra from IAVs showed that the corresponding IAVs subtypes could be distinguished in 90 min, accurately. Furthermore, the principal component analysis results also show that nine virus subtypes can be reliably distinguished. More importantly, this strategy provides an alternative method for identifying and distinguishing other viruses with high variability characteristics, such as SARS-CoV-2, which could be helpful for implementing public health strategies to counter pandemics. A MALDI-TOF MS based universal strategy for the discrimination of virus subtypes was developed, which possess the advantages of speed and high-accuracy. [Display omitted] • A home-made identification database of influenza A virus subtypes was set up. • The discrimination of influenza A virus subtypes could be finished within 90 min. • The influenza A virus subtypes could be distinguished with high accuracy. [ABSTRACT FROM AUTHOR]

Following its spread in the USA, West Nile Virus (WNV) has reemerged in the Mediterranean basin with a renewed pathogenicity. The introduction of WNV lineage 2 in Europe and its co-circulation with lineage 1 has resulted in a continuously changing epidemiological scenario, highlighting the importance of differential detection of the two lineages. The paper describes a new real-time PCR method for the detection and genotyping of the two main lineages of WNV. The method requires a single pair of primers and probes and is based on the analysis of highly conserved consensus sequences detected in the 5' terminus of the viral genome. [ABSTRACT FROM AUTHOR]

Aim Few studies have examined the effect of combined low-risk human papillomavirus ( LR-HPV) and high-risk human papillomavirus ( HR-HPV) infection on the progression of cervical intraepithelial neoplasia ( CIN)2 to CIN3. This multi-institutional prospective cohort study investigated the risk of progression of CIN2 with various combinations of HR-HPV and LR-HPV infection. Methods Between January 2007 and May 2008, 122 women with CIN2 (aged 20-50 years) from 24 hospitals throughout Japan were enrolled in the study. Ninety-three women were analyzed after a 2-year follow-up with cytology, colposcopy, HR-HPV testing and HPV genotyping. Colposcopy-directed biopsy was performed at entry and the end of this study, or when disease progression was suspected. Results Among 93 women with CIN2, 87 (93.5%) had HR-HPV infection. Among these 87 cases, 24 (27.6%) progressed to CIN3 and 49 (56.3%) regressed. None of the six women with CIN2 without HR-HPV infection progressed. The progression rate was significantly lower in women with combined HR-HPV and LR-HPV infection (3/28, 10.7%) than in those with HR-HPV infection only (21/59, 35.6%; P = 0.016). Multivariate analyses showed that CIN2 progression in women with HR-HPV infection was negatively associated with LR-HPV co-infection (hazard ratio = 0.152; 95% confidence interval [ CI] = 0.042-0.553). CIN2 regression was positively associated with LR-HPV co-infection (odds ratio = 4.553; 95% CI = 1.378-15.039). Conclusion The risk of CIN2 progression is low in women with combined infection of HR-HPV and LR-HPV. The finding may be useful for management of women diagnosed with CIN2. [ABSTRACT FROM AUTHOR]

Abstract: A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide–Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through “sandwich” hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin–tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/μL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence. [Copyright &y& Elsevier]

Abstract: In this study, a novel microfluidic device with microbead array was developed and sensitive genotyping of HBV was demonstrated using quantum dot as labels. This device was assembled by using two PDMS slabs featured with different microstructures and channel depths for the construction of a functional region comprising a chamber array and a single sampling microchannel. Since the chamber array and its sampling channel are of different channel depths and are bonded face-to-face, weir structures are generated to confine the microbeads which could be addressed using the microfluidic channel. Highly sensitive virus DNA detection was achieved by the enhanced mass transport in the microfluidics and the rapid reaction dynamics of suspension microbead array. The device could detect 1000copies/mL of HBV virus in clinical serum samples using in vitro transcribed RNA as the target molecules. Based on DNA hybridization with quantum dots labels, on-chip virus genotyping was also demonstrated with high discrimination specificity and sensitivity (4pM, S/N >3) using synthesized HBV DNA probes. This microfluidic device combines the rapid binding kinetics of homogeneous assays of microbead array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for high sensitivity virus DNA analysis with small reagent consumption, short assay time and parallel detection. [Copyright &y& Elsevier]