A resampling strategy for studying robustness in virus detection pipelines

Next-generation sequencing is regularly used to identify viral sequences in DNA or RNA samples of infected hosts. A major step of most pipelines for virus detection is to map sequence reads against known virus genomes. Due to small differences between the sequences of related viruses, and due to several biological or technical errors, mapping underlies uncertainties. As a consequence, the resulting list of detected viruses can lack robustness.A new approach for generating artificial sequencing reads together with a strategy of resampling from the original findings is proposed that can help to assess the robustness of the originally identified list of viruses. From the original mapping result in form of a SAM file, a set of statistical distributions are derived. These are used in the resampling pipeline to generate new artificial reads which are again mapped versus the reference genomes. By summarizing the resampling procedure, the analyst receives information about whether the presence of a particular virus in the sample gains or losses evidence, and thus about the robustness of the original mapping list but also that of individual viruses in this list. To judge robustness, several indicators are derived from the resampling procedure such as the correlation between original and resampling read counts, or the statistical detection of outliers in the differences of read counts. Additionally, graphical illustrations of read count shifts via Sankey diagrams are provided.To demonstrate the use of the new approach, the resampling approach is applied to three real-world data samples, one of them with laboratory-confirmed Influenza sequences, and to artificially generated data where virus sequences have been spiked into the sequencing data of a host. By applying the resampling pipeline, several viruses drop from the original list while new viruses emerge, showing robustness of those viruses that remain in the list.The evaluation of the new approach shows that the resampling approach is helpful to analyze the viral content of a biological sample, to rate the robustness of original findings and to better show the overall distribution of findings. The method is also applicable to other virus detection pipelines based on read mapping.     

Recovery of known T-cell epitopes by computational scanning of a viral genome

A new computational method (EpiDock) is proposed for predicting peptide binding to class I MHC proteins, from the amino acid sequence of any protein of immunological interest. Starting from the primary structure of the target protein, individual three-dimensional structures of all possible MHC-peptide (8-, 9- and 10-mers) complexes are obtained by homology modelling. A free energy scoring function (Fresno) is then used to predict the absolute binding free energy of all possible peptides to the class I MHC restriction protein. Assuming that immunodominant epitopes are usually found among the top MHC binders, the method can thus be applied to predict the location of immunogenic peptides on the sequence of the protein target. When applied to the prediction of HLA-A^*0201-restricted T-cell epitopes from the Hepatitis B virus, EpiDock was able to recover 92% of known high affinity binders and 80% of known epitopes within a filtered subset of all possible nonapeptides corresponding to about one tenth of the full theoretical list.The proposed method is fully automated and fast enough to scan a viral genome in less than an hour on a parallel computing architecture. As it requires very few starting experimental data, EpiDock can be used: (i) to predict potential T-cell epitopes from viral genomes (ii) to roughly predict still unknown peptide binding motifs for novel class I MHC alleles.     

Mutational spectrum of SARS-CoV-2 during the global pandemic

Viruses accumulate mutations under the influence of natural selection and host–virus interactions. Through a systematic comparison of 351,525 full viral genome sequences collected during the recent COVID-19 pandemic, we reveal the spectrum of SARS-CoV-2 mutations. Unlike those of other viruses, the mutational spectrum of SARS-CoV-2 exhibits extreme asymmetry, with a much higher rate of C>U than U>C substitutions, as well as a higher rate of G>U than U>G substitutions. This suggests directional genome sequence evolution during transmission. The substantial asymmetry and directionality of the mutational spectrum enable pseudotemporal tracing of SARS-CoV-2 without prior information about the root sequence, collection time, and sampling region. This shows that the viral genome sequences collected in Asia are similar to the original genome sequence. Adjusted estimation of the dN/dS ratio accounting for the asymmetrical mutational spectrum also shows evidence of negative selection on viral genes, consistent with previous reports. Our findings provide deep insights into the mutational processes in SARS-CoV-2 viral infection and advance the understanding of the history and future evolution of the virus.Subject terms: Evolutionary biology, Genome evolution     

Photochemical inactivation of alpha- and poxviruses

The objective of this study was to determine whether photochemical inactivation of viruses could be accomplished with high efficiency while preserving the molecular integrity of viral targets allowing subsequent diagnostic tests to be performed at a lower level of containment and cost. We studied the effect of 5-iodonaphthyl 1-azide (INA) and amotosalen (AMO, also known as S-59), which are photochemicals known to target either viral proteins or nucleic acids, respectively. We found that vaccinia virus (VACV, an orthopox virus with a DNA genome) and pixuna virus (PIXV, an alphavirus with an RNA genome) were stable when irradiated with UVA alone or when exposed to either INA or AMO in the dark. AMO followed by UVA exposure was at least 1000-fold more virucidal than INA/UVA on vaccinia and pixuna viruses treated under similar conditions. Photoinactivation with either INA or AMO at conditions that abolished viral infectivity resulted in only minimal impairment of subsequent ELISA and PCR testing. The results presented in this study should assist in developing methods to inactivate in the field environmental and forensic samples suspected of viral contamination, thus limiting the need for costly security and safety operations after an accidental or intentional viral release.     

Mechanical characterization of HIV‐1 with a solid‐state nanopore sensor

Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo‐type human immunodeficiency virus type 1 at sub‐micron scale. Non‐infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.     

DIFFERENTIAL EFFECTS OF PHOTOACTIVE FURANYL COMPOUNDS ON VIRUS FUNCTIONS

Abstract— Five photoactive furanyl compounds were investigated for their activities against viruses. The two furanocoumarins used were 8-methoxypsoralen (8-MOP) and angelicin; two furanochromones, visnagin and khellin, and the furanoquinoline, dictamnine, were also used. The DNA-containing herpes virus murine cytomegalovirus (MCMV) and the RNA-containing togavirus, Sindbis virus, were the target viruses. All five compounds inactivated both viruses in the presence of UVA, although Sindbis virus was much less sensitive. The relative order of antiviral potency was 8-MOP > dictamnine > visnagin > angelicin > khellin. Dictamnine however was slightly more effective than 8-MOP against Sindbis virus. None of the treatments affected the structural integrity of MCMV, nor did they interfere with the normal transit of the virus into host cells or the localisation of the viral genome in the cell nucleus. Some early viral gene functions were expressed but the viruses did not replicate.     

Unraveling virus identity by detection of depleted probes with capillary electrophoresis

With the emergence of new viral infections and pandemics, there is a need to develop faster methods to unravel the virus identities in a large number of clinical samples. This report describes a virus identification method featuring high throughput, high resolution, and high sensitivity detection of viruses. Identification of virus is based on liquid hybridization of different lengths of virus-specific probes to their corresponding viruses. The probes bound to target sequences are removed by a biotin–streptavidin pull-down mechanism and the supernatant is analyzed by capillary electrophoresis. The probes depleted from the sample appear as diminished peaks in the electropherograms and the remaining probes serve as calibrators to align peaks in different capillaries. The virus identities are unraveled by a signal processing and peak detection algorithm developed in-house. Nine viruses were used in the study to demonstrate how the system works to unravel the virus identity in single and double virus infections. With properly designed probes, the system is able to distinguish closely related viruses. The system takes advantage of the high resolution feature of capillary electrophoresis to resolve probes that differ by length. The method may facilitate virus identity screen from more candidate viruses with an automated 4-color DNA sequencer.     

Affinity-Based Screening Technology and HCV Drug Discovery

NS5A is one of the non-structural gene products encoded by Hepatitis C virus (HCV) and related viruses that are essential for viral replication. The amino acid sequence of NS5A is conserved between different HCV genotypes and the primary amino acid sequence of NS5A is unique to HCV and closely related viruses. Importantly, NS5A is unrelated to any human protein. This indicates that drugs designed to block the actions of NS5A could inhibit the replication of HCV without showing toxic side effects in human host cells, thus making NS5A inhibitors ideal anti-viral drugs. However, there are presently no functional assays for this essential viral protein. Therefore, conventional high throughput screening (HTS) approaches can not be used to discover antiviral drugs against NS5A.     

Predicting interspecies transmission of avian influenza virus based on wavelet packet decomposition

Using wavelet packet decomposition, the energy coefficients in the fifth level of viral protein sequences were achieved to predict interspecies transmission. Since avian-origin influenza viruses could have high sequence similarities with human-origin avian influenza virus and could have the phenotype of interspecies transmission, viral data should be filtered to prevent the misconduct of feature selection and false performance of predicting models. Considering the balance of data size, the empirical cut-off value 97% was used to screen avian-origin influenza virus with high sequence similarity. The excellent performances of cross validation show that the SVM model has the best capability of predicting transmission and evaluating the contribution of five amino acid factors. The robust model was finally used to evaluate the filtered data of avian-origin virus and the results confirmed that double check for ambiguous phenotype of avian-origin virus with high sequence similarity was necessary and part of them have the ability to across species barriers.     

Viral-induced self-assembly of magnetic nanoparticles allows the detection of viral particles in biological media

Monodisperse magnetic nanoparticles conjugated with virus-surface-specific antibodies self-assemble in the presence of specific viral particles to create supramolecular structures with enhanced magnetic properties, as detected by magnetic resonance methods (NMR/MRI). The observed magnetic relaxation changes that occur upon viral-induced assembly allowed for highly sensitive and selective detection of a virus in complex biological media. The developed method was shown to specifically detect adenovirus-5 and herpes simplex virus-1 at concentrations of 5 viral particles/10 muL without the need of extensive sample preparation. The applications of this new method span from high-throughput NMR detection of viruses in biological samples to potential MR imaging of viral distribution in vivo.     

Removal of poliovirus type 1 from a protein mixture using an immunoaffinity chromatography column.

An immunoaffinity matrix was prepared using human polyclonal antibody (Intragam) attached to Sepharose 4B activated with CNBr. The immunoaffinity matrix was then assessed with regard to its capacity to remove viruses. The challenge virus, poliovirus type 1 was loaded in high titre in either PBS or a preparation derived from human plasma known as supernatant II + III. This fraction is depleted of IgG and is used to prepare human albumin. It was shown that an average greater than 5 logs of spiked virus were removed in one passage through the column. This type of approach may prove useful as a viral removal method in biopharmaceutical manufacturing.     

Activation, exposure and penetration of virally encoded, membrane-active polypeptides during non-enveloped virus entry

Host cell entry by influenza and other enveloped viruses is well characterized, however, the manner in which non-enveloped viruses deliver their genome across host cell membranes in the absence of membrane fusion remains unresolved. The discovery of short, membrane altering, amphipathic or hydrophobic sequences in several non-enveloped virus capsid proteins such as the gamma (gamma) peptide of nodaviruses and tetraviruses, VP4 and the N-terminal region of VP1 of picornaviruses, micro1N of reoviruses, and protein VI of adenoviruses suggests that these small peptides facilitate breaching of the host membrane and the delivery of the viral genome into the host cell. In spite of conspicuous differences in entry among non-enveloped virions, the short stretches of membrane active regions are associated with similar, entry-related events including: I) proteolytic cleavage of a precursor capsid protein resulting in increased dynamic character and/or accessibility of these peptides; II) structural changes in the virus capsid triggered by receptor binding and/or low pH in entry compartments, resulting in peptide exposure; III) externalized peptides interact with host membranes and disrupt them, facilitating delivery of the viral genome inside the host cell. Here we discuss the membrane alteration activity in non-enveloped viruses with reference to the gamma peptide of nodaviruses. Virtually all of the characteristics of gamma are shared by analogous peptides in other non-enveloped viruses, making it a simple prototype for comparative purposes.     

Mass spectrometry based proteomic studies on viruses and hosts--a review

In terms of proteomic research in the 21st century, the realm of virology is still regarded as an enormous challenge mainly brought by three aspects, namely, studying on the complex proteome of the virus with unexpected variations, developing more accurate analytical techniques as well as understanding viral pathogenesis and virus–host interaction dynamics. Progresses in these areas will be helpful to vaccine design and antiviral drugs discovery. Mass spectrometry based proteomics have shown exceptional display of capabilities, not only precisely identifying viral and cellular proteins that are functionally, structurally, and dynamically changed upon virus infection, but also enabling us to detect important pathway proteins. In addition, many isolation and purification techniques and quantitative strategies in conjunction with MS can significantly improve the sensitivity of mass spectrometry for detecting low-abundant proteins, replenishing the stock of virus proteome and enlarging the protein–protein interaction maps. Nevertheless, only a small proportion of the infectious viruses in both of animal and plant have been studied using this approach. As more virus and host genomes are being sequenced, MS-based proteomics is becoming an indispensable tool for virology. In this paper, we provide a brief review of the current technologies and their applications in studying selected viruses and hosts.     

Molecular characterization of pigeon torque teno virus (PTTV) in Jiangsu province

The torque teno virus (TTV) is a recently discovered DNA virus that has been detected in many different hosts, including humans, livestock and poultry. To date, there is no report of pigeon TTV (PTTV) from anywhere in the world. To investigate the distribution of PTTV in pigeons from the eastern Chinese province of Jiangsu and characterize their genomes, we employed PCR to detect PTTV in 144 samples collected from 6 pigeon plants in Jiangsu province, amplify complete genomes from representative samples and analyze genetic characteristics using bioinformatics. The results demonstrated that 71.5% (103/144) of samples were PTTV positive. The rate of sequence homology among the six PTTV complete genomes obtained from Jiangsu province ranged from 99.7% to 100%. Phylogenetic analysis suggested that PTTV genomes had a high degree of genetic similarity and were similar to chicken anemia virus that also had poultry as a host. Although with the same host, PTTV shared distant relationship with PiCV in both complete genome, Rep and Cap genes. The results of this study provided evidence that PTTV could be detected in Chinese pigeons at a high level, the evolutionary process of complete genome, Rep and Cap genes of Anelloviridae family had obvious divergence.     

Capillary electrophoresis of viruses, subviral particles and virus complexes

CZE and CIEF were so far applied to the analysis of tobacco mosaic virus, Semliki forest virus, human rhinovirus, adenovirus, norovirus and the bacteriophages T5 and MS2. The concentration of viral or subviral particles, of capsid proteins and viral genomes were determined, their electrophoretic mobilities and pI values were measured and bioaffinity reactions between viruses and antibodies, antibody fragments and receptor fragments were assessed. The role of detergents added to the BGE to obtain reproducible electrophoretic conditions was elucidated. The analytes were detected via their UV-absorbance or via fluorescence after derivatization of the viral capsid, the nucleic acid, or both. A new dimension to the detection is added by the possibility of making use of the viral infectivity. At least in theory, this allows for the unequivocal identification of a single infectious virus particle after collection at the capillary outlet. This review summarizes the 25 papers so far published on this topic.     

单病毒示踪

病毒是对人类健康威胁最大的病原之一,由其引发的病毒性疾病对人民健康、国家安全和社会经济构成重大威胁.病毒感染机制研究对病毒性疾病的防控及治疗具有重大意义.病毒侵染宿主细胞的动态过程涉及病毒组分与多种细胞组分或细胞器间复杂的相互作用,但是传统手段无法对该动态过程进行实时跟踪研究.单病毒示踪技术作为一种可以实时原位示踪单颗...