HTS-Based Diagnostics of Sugarcane Viruses: Seasonal Variation and Its Implications for Accurate Detection

Rapid global germplasm trade has increased concern about the spread of plant pathogens and pests across borders that could become established, affecting agriculture and environment systems. Viral pathogens are of particular concern due to their difficulty to control once established. A comprehensive diagnostic platform that accurately detects both known and unknown virus species, as well as unreported variants, is playing a pivotal role across plant germplasm quarantine programs. Here we propose the addition of high-throughput sequencing (HTS) from total RNA to the routine quarantine diagnostic workflow of sugarcane viruses. We evaluated the impact of sequencing depth needed for the HTS-based identification of seven regulated sugarcane RNA/DNA viruses across two different growing seasons (spring and fall). Our HTS analysis revealed that viral normalized read counts (RPKM) was up to 23-times higher in spring than in the fall season for six out of the seven viruses. Random read subsampling analyses suggested that the minimum number of reads required for reliable detection of RNA viruses was 0.5 million, with a viral genome coverage of at least 92%. Using an HTS-based total RNA metagenomics approach, we identified all targeted viruses independent of the time of the year, highlighting that higher sequencing depth is needed for the identification of DNA viruses.     

Performance characterization of PCR-free whole genome sequencing for clinical diagnosis

To evaluate the performance of polymerase chain reaction (PCR)-free whole genome sequencing (WGS) for clinical diagnosis, and thereby revealing how experimental parameters affect variant detection.Five NA12878 samples were sequenced using MGISEQ-2000. NA12878 samples underwent WGS with differing deoxyribonucleic acid (DNA) input and library preparation protocol (PCR-based vs PCR-free protocols for library preparation). The depth of coverage and genotype quality of each sample were compared. The performance of each sample was measured for sensitivity, coverage of depth and breadth of coverage of disease-related genes, and copy number variants. We also developed a systematic WGS pipeline (PCR-free) for the analysis of 11 clinical cases.In general, NA12878-2 (PCR-free WGS) showed better depth of coverage and genotype quality distribution than NA12878-1 (PCR-based WGS). With a mean depth of ∼40×, the sensitivity of homozygous and heterozygous single nucleotide polymorphisms (SNPs) of NA12878-2 showed higher sensitivity (>99.77% and >99.82%) than NA12878-1, and positive predictive value exceeded 99.98% and 99.07%. The sensitivity and positive predictive value of homozygous and heterozygous indels for NA12878-2 (PCR-free WGS) showed great improvement than NA128878-1. The breadths of coverage for disease-related genes and copy number variants are slightly better for samples with PCR-free library preparation protocol than the sample with PCR-based library preparation protocol. DNA input also influences the performance of variant detection in samples with PCR-free WGS. All the 19 previously confirmed variants in 11 clinical cases were successfully detected by our WGS pipeline (PCR free).Different experimental parameters may affect variant detection for clinical WGS. Clinical scientists should know the range of sensitivity of variants for different methods of WGS, which would be useful when interpreting and delivering clinical reports.     

Utility of a Sequence-Independent,Single-Primer-Amplification (SISPA) and Nanopore Sequencing Approach for Detection and Characterization of Tick-Borne Viral Pathogens

Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA with identities over 99%. Genome coverage varied according to the segment and total read count. Base calling errors were mainly observed in tick specimens and more frequent in lower viral loads. Culture-grown viruses were phylogenetically-related to previously-reported local viruses. In conclusion, the SISPA + nanopore sequencing was successful in generating data comparable to NGS and will provide an effective tool for broad-range virus detection in ticks.     

LABRADOR—A Computational Workflow for Virus Detection in High-Throughput Sequencing Data

High-throughput sequencing (HTS) allows detection of known and unknown viruses in samples of broad origin. This makes HTS a perfect technology to determine whether or not the biological products, such as vaccines are free from the adventitious agents, which could support or replace extensive testing using various in vitro and in vivo assays. Due to bioinformatics complexities, there is a need for standardized and reliable methods to manage HTS generated data in this field. Thus, we developed LABRADOR—an analysis pipeline for adventitious virus detection. The pipeline consists of several third-party programs and is divided into two major parts: (i) direct reads classification based on the comparison of characteristic profiles between reads and sequences deposited in the database supported with alignment of to the best matching reference sequence and (ii) de novo assembly of contigs and their classification on nucleotide and amino acid levels. To meet the requirements published in guidelines for biologicals’ safety we generated a custom nucleotide database with viral sequences. We tested our pipeline on publicly available HTS datasets and showed that LABRADOR can reliably detect viruses in mixtures of model viruses, vaccines and clinical samples.     

Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.     

Single Amino Acid Mutations Affect Zika Virus Replication In Vitro and Virulence In Vivo

The 2014–2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.     

Walking Together: Cross-Protection,Genome Conservation,and the Replication Machinery of Citrus tristeza virus

“Cross-protection”, a nearly 100 years-old virological term, is suggested to be changed to “close protection”. Evidence for the need of such change has accumulated over the past six decades from the laboratory experiments and field tests conducted by plant pathologists and plant virologists working with different plant viruses, and, in particular, from research on Citrus tristeza virus (CTV). A direct confirmation of such close protection came with the finding that “pre-immunization” of citrus plants with the variants of the T36 strain of CTV but not with variants of other virus strains was providing protection against a fluorescent protein-tagged T36-based recombinant virus variant. Under natural conditions close protection is functional and is closely associated both with the conservation of the CTV genome sequence and prevention of superinfection by closely similar isolates. It is suggested that the mechanism is primarily directed to prevent the danger of virus population collapse that could be expected to result through quasispecies divergence of large RNA genomes of the CTV variants continuously replicating within long-living and highly voluminous fruit trees. This review article provides an overview of the CTV cross-protection research, along with a discussion of the phenomenon in the context of the CTV biology and genetics.     

Diagnostic tests for hepatitis C: Recent trends in electrochemical immunosensor and genosensor analysis

Hepatitis C is a liver disease that is transmitted through contact with the blood of an infected person. An estimated 150 million individuals worldwide have been chronically infected with the hepatitis C virus (HCV). Hepatitis C shows significant genetic variation in the global population, due to the high rate of viral RNA mutation. There are six variants of the virus (HCV genotypes 1, 2, 3, 4, 5, and 6), with 15 recorded subtypes that vary in prevalence across different regions of the world. A variety of devices are used to diagnose hepatitis C, including HCV antibody test, HCV viral load test, HCV genotype test and liver biopsy. Rapid, inexpensive, sensitive, and robust analytical devices are therefore essential for effective diagnosis and monitoring of disease treatment. This review provides an overview of current electrochemical immunosensor and genosensor technologies employed in HCV detection. There are a limited number of publications showing electrochemical biosensors being used for the detection of HCV. Due to their simplicity, specificity, and reliability, electrochemical biosensor devices have potential clinical applications in several viral infections.     

DsRNA Sequencing for RNA Virus Surveillance Using Human Clinical Samples

Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.     

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.     

On-Demand Patient-Specific Phenotype-to-Genotype Ebola Virus Characterization

Biosafety, biosecurity, logistical, political, and technical considerations can delay or prevent the wide dissemination of source material containing viable virus from the geographic origin of an outbreak to laboratories involved in developing medical countermeasures (MCMs). However, once virus genome sequence information is available from clinical samples, reverse-genetics systems can be used to generate virus stocks de novo to initiate MCM development. In this study, we developed a reverse-genetics system for natural isolates of Ebola virus (EBOV) variants Makona, Tumba, and Ituri, which have been challenging to obtain. These systems were generated starting solely with in silico genome sequence information and have been used successfully to produce recombinant stocks of each of the viruses for use in MCM testing. The antiviral activity of MCMs targeting viral entry varied depending on the recombinant virus isolate used. Collectively, selecting and synthetically engineering emerging EBOV variants and demonstrating their efficacy against available MCMs will be crucial for answering pressing public health and biosecurity concerns during Ebola disease (EBOD) outbreaks.     

Molecular and serological characterization of hepatitis B virus in deferred Ghanaian blood donors with and without elevated alanine aminotransferase

Candidate blood donors in Ghana are frequent carriers of hepatitis B virus (HBV). A comparative study of 117 donor samples including 46 with alanine aminotransferase (ALT) > or = 60 IU/L and 71 with < or =40 IU/L level was undertaken. S and the basic core promoter-precore regions (BCP/PC) sequencing was used to identify genotypes and variants relevant to HBV natural history, respectively. Age, viral load, HBe status were correlated with molecular data. HBV genotype E (87%) was dominant with little genotypes A (10%) and D (3%). Comparing individuals with or without liver disease, an association between liver disease and older age (P = 0.004) and higher viral load (P = 0.002) whether as a whole population or only genotype E was found. Compared with a commercial assay, BCP/PC sequencing had lower sensitivity to detect mixtures of wild-type and variant viruses but detected BCP deletions. BCP 1762/1764 variants were positively correlated with older age (P < 0.0001) and elevated ALT levels (P = 0.01). PC 1896 stop codon was marginally correlated with viral load (P = 0.09). HBV genotype E infection natural history appears different from genotypes B and C prevalent in Asia. Donors with liver disease being older, with higher viral load and higher BCP variant proportion may be at higher risk of cirrhosis and hepatocellular carcinoma.     

Targeted Genome Sequencing (TG-Seq) Approaches to Detect Plant Viruses

Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of; Pea early browning virus (PEBV), Cucumber mosaic virus (CMV), Bean yellow mosaic virus (BYMV) and Pea seedborne mosaic virus (PSbMV). TG-Seq detected all the expected viral amplicons within multiplex PCR (mPCR) reactions. In contrast, the expected PCR amplicons were not detected by gel electrophoresis (GE). For example, for CMV, GE only detected RNA1 and RNA2 while TG-Seq detected all the three RNA components of CMV. In an mPCR to amplify all four viruses, TG-Seq readily detected each virus with more than 732,277 sequence reads mapping to each amplicon. In addition, TG-Seq also detected all four amplicons within a 10⁻⁸ serial dilution that were not detectable by GE. Our current findings reveal that the TG-Seq approach offers significant potential and is a highly sensitive targeted approach for detecting multiple plant viruses within a given biological sample. This is the first study describing direct HTS of plant virus mPCR products. These findings have major implications for grain germplasm healthy certification programs and biosecurity management in relation to pathogen entry into Australia and elsewhere.     

Identification of Highly Conserved SARS-CoV-2 Antigenic Epitopes with Wide Coverage Using Reverse Vaccinology Approach

One of the most effective strategies for eliminating new and emerging infectious diseases is effective immunization. The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) warrants the need for a maximum coverage vaccine. Moreover, mutations that arise within the virus have a significant impact on the vaccination strategy. Here, we built a comprehensive in silico workflow pipeline to identify B-cell- and T-cell-stimulating antigens of SARS-CoV-2 viral proteins. Our in silico reverse vaccinology (RV) approach consisted of two parts: (1) analysis of the selected viral proteins based on annotated cellular location, antigenicity, allele coverage, epitope density, and mutation density and (2) analysis of the various aspects of the epitopes, including antigenicity, allele coverage, IFN-γ induction, toxicity, host homology, and site mutational density. After performing a mutation analysis based on the contemporary mutational amino acid substitutions observed in the viral variants, 13 potential epitopes were selected as subunit vaccine candidates. Despite mutational amino acid substitutions, most epitope sequences were predicted to retain immunogenicity without toxicity and host homology. Our RV approach using an in silico pipeline may potentially reduce the time required for effective vaccine development and can be applicable for vaccine development for other pathogenic diseases as well.     

SARS-CoV-2: Evolution and Emergence of New Viral Variants

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the coronavirus disease 2019 (COVID-19). The high rate of mutation of this virus is associated with a quick emergence of new viral variants that have been rapidly spreading worldwide. Several mutations have been documented in the receptor-binding domain (RBD) of the viral spike protein that increases the interaction between SARS-CoV-2 and its cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Mutations in the spike can increase the viral spread rate, disease severity, and the ability of the virus to evade either the immune protective responses, monoclonal antibody treatments, or the efficacy of current licensed vaccines. This review aimed to highlight the functional virus classification used by the World Health Organization (WHO), Phylogenetic Assignment of Named Global Outbreak (PANGO), Global Initiative on Sharing All Influenza Data (GISAID), and Nextstrain, an open-source project to harness the scientific and public health potential of pathogen genome data, the chronological emergence of viral variants of concern (VOCs) and variants of interest (VOIs), the major findings related to the rate of spread, and the mutations in the spike protein that are involved in the evasion of the host immune responses elicited by prior SARS-CoV-2 infections and by the protection induced by vaccination.     

Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics

Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.