VIRify: An integrated detection, annotation and taxonomic classification pipeline using virus-specific protein profile hidden Markov models.

The study of viral communities has revealed the enormous diversity and impact these biological entities have on various ecosystems. These observations have sparked widespread interest in developing computational strategies that support the comprehensive characterisation of viral communities based on sequencing data. Here we introduce VIRify, a new computational pipeline designed to provide a user-friendly and accurate functional and taxonomic characterisation of viral communities. VIRify identifies viral contigs and prophages from metagenomic assemblies and annotates them using a collection of viral profile hidden Markov models (HMMs). These include our manually-curated profile HMMs, which serve as specific taxonomic markers for a wide range of prokaryotic and eukaryotic viral taxa and are thus used to reliably classify viral contigs. We tested VIRify on assemblies from two microbial mock communities, a large metagenomics study, and a collection of publicly available viral genomic sequences from the human gut. The results showed that VIRify could identify sequences from both prokaryotic and eukaryotic viruses, and provided taxonomic classifications from the genus to the family rank with an average accuracy of 86.6%. In addition, VIRify allowed the detection and taxonomic classification of a range of prokaryotic and eukaryotic viruses present in 243 marine metagenomic assemblies. Finally, the use of VIRify led to a large expansion in the number of taxonomically classified human gut viral sequences and the improvement of outdated and shallow taxonomic classifications. Overall, we demonstrate that VIRify is a novel and powerful resource that offers an enhanced capability to detect a broad range of viral contigs and taxonomically classify them.

Receptor-Binding-Motif-Targeted Sanger Sequencing: a Quick and Cost-Effective Strategy for Molecular Surveillance of SARS-CoV-2 Variants.

Whole-genome sequencing (WGS) is the gold standard for characterizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and identification of new variants. However, the cost involved and time needed for WGS prevent routine, rapid clinical use. This study aimed to develop a quick and cost-effective surveillance strategy for SARS-CoV-2 variants in saliva and nasal swab samples by spike protein receptor-binding-motif (RBM)-targeted Sanger sequencing. Saliva and nasal swabs prescreened for the presence of the nucleocapsid (N) gene of SARS-CoV-2 were subjected to RBM-specific single-amplicon generation and Sanger sequencing. Sequences were aligned by CLC Sequence Viewer 8, and variants were identified based upon specific mutation signature. Based on this strategy, the present study identified Alpha, Beta/Gamma, Delta, and Omicron variants in a quick and cost-effective manner. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns. Following the Alpha variant (B.1.1.7) infection wave, the Delta variant (B.1.617.2) became prevalent, and now the recently identified Omicron (B.1.1.529) variant is spreading rapidly and forming BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages of concern. Prompt identification of mutational changes in SARS-CoV-2 variants is challenging but critical to managing the disease spread and vaccine/therapeutic modifications. Considering the cost involved and resource limitation of WGS globally, an RBM-targeted Sanger sequencing strategy is adopted in this study for quick molecular surveillance of SARS-CoV-2 variants.

Assessing in vivo mutation frequencies and creating a high-resolution genome-wide map of fitness costs of Hepatitis C virus.

Like many viruses, Hepatitis C Virus (HCV) has a high mutation rate, which helps the virus adapt quickly, but mutations come with fitness costs. Fitness costs can be studied by different approaches, such as experimental or frequency-based approaches. The frequency-based approach is particularly useful to estimate in vivo fitness costs, but this approach works best with deep sequencing data from many hosts are. In this study, we applied the frequency-based approach to a large dataset of 195 patients and estimated the fitness costs of mutations at 7957 sites along the HCV genome. We used beta regression and random forest models to better understand how different factors influenced fitness costs. Our results revealed that costs of nonsynonymous mutations were three times higher than those of synonymous mutations, and mutations at nucleotides A or T had higher costs than those at C or G. Genome location had a modest effect, with lower costs for mutations in HVR1 and higher costs for mutations in Core and NS5B. Resistance mutations were, on average, costlier than other mutations. Our results show that in vivo fitness costs of mutations can be site and virus specific, reinforcing the utility of constructing in vivo fitness cost maps of viral genomes.

Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission.

Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer 2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16-20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement.

Comparative Assessment of Bacteriophage and Antibiotic Activity against Multidrug-Resistant Staphylococcus aureus Biofilms.

Problems connected with biofilm-related infections and antibiotic resistance necessitate the investigation and development of novel treatment strategies. Given their unique characteristics, one of the most promising alternatives to conventional antibiotics are bacteriophages. In the in vitro and in vivo larva model study, we demonstrate that phages vB_SauM-A, vB_SauM-C, and vB_SauM-D are effective antibiofilm agents. The exposure of biofilm to phages vB_SauM-A and vB_SauM-D led to 2-3 log reductions in the colony-forming unit number in most of the multidrug-resistant S. aureus strains. It was found that phage application reduced the formed biofilms independently of the used titer. Moreover, the study demonstrated that bacteriophages are more efficient in biofilm biomass removal and reduction in staphylococci count when compared to the antibiotics used. The scanning electron microscopy analysis results are in line with colony forming unit (CFU) counting but not entirely consistent with crystal violet (CV) staining. Additionally, phages vB_SauM-A, vB_SauM-C, and vB_SauM-D can significantly increase the survival rate and extend the survival time of Galleria mellonella larvae.

Assessment of two-pool multiplex long-amplicon nanopore sequencing of SARS-CoV-2.

Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in COVID-19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS-CoV-2 followed by either massively parallel or nanopore sequencing is a widely-used strategy to obtain genome sequences from primary samples. However, multiplex PCR-based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two-pool, long-amplicon 36-plex PCR primer panel with ~1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2. We validated the panel by assessing nasopharyngeal swab samples with a <30 quantitative reverse transcription PCR cycle threshold value and found that ≥90% of viral genomes could be covered with high sequencing depths (≥20% mean depth). In comparison, the widely-used ARTIC panel yielded 79%-88% high-depth genome regions. We estimated that ~5 Mbp nanopore sequencing data may ensure a >95% viral genome coverage with a ≥10-fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false-positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5' or 3' ends of reads. Thus, nanopore sequencing could not elucidate intra-host viral diversity.

Assessment of mycoviral diversity in Pakistani fungal isolates revealed infection by 11 novel viruses of a single strain of Fusarium mangiferae isolate SP1.

An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

In Vitro Characterization and In Vivo Efficacy Assessment in Galleria mellonella Larvae of Newly Isolated Bacteriophages against Escherichia coli K1.

Extra-intestinal Escherichia coli express several virulence factors that increase their ability to colonize and survive in different localizations. The K1 capsular type is involved in several infections, including meningitis, urinary tract, and bloodstream infections. The aims of this work were to isolate, characterize, and assess the in vivo efficacy of phages targeting avian pathogenic E. coli (APEC) O18:K1, which shares many similarities with the human strains responsible for neonatal meningitis. Eleven phages were isolated against APEC O18:K1, and four of them presenting a narrow spectrum targeting E. coli K1 strains were further studied. The newly isolated phages vB_EcoS_K1-ULINTec2 were similar to the Siphoviridae family, and vB_EcoP_K1-ULINTec4, vB_EcoP_K1-ULINTec6, and vB_EcoP_K1-ULINTec7 to the Autographiviridae family. They are capsular type (K1) dependent and present several advantages characteristic of lytic phages, such as a short adsorption time and latent period. vB_EcoP_K1-ULINTec7 is able to target both K1 and K5 strains. This study shows that these phages replicate efficiently, both in vitro and in vivo in the Galleria mellonella model. Phage treatment increases the larvae survival rates, even though none of the phages were able to eliminate the bacterial load.

Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol.

Fast and effective methods are needed for sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome to track genetic mutations and to identify new and emerging variants during the ongoing pandemic. The objectives were to assess the performance of the SARS-CoV-2 AmpliSeq research panel and S5 plug-in analysis tools for whole-genome sequencing analysis of SARS-CoV-2 and to compare the results with those obtained with the MiSeq-based ARTIC analysis pipeline, using metrics such as depth, coverage, and concordance of single-nucleotide variant (SNV) calls. A total of 191 clinical specimens and a single cultured isolate were extracted and sequenced with AmpliSeq technology and analysis tools. Of the 191 clinical specimens, 83 (with threshold cycle [CT] values of 15.58 to 32.54) were also sequenced using an Illumina MiSeq-based method with the ARTIC analysis pipeline, for direct comparison. A total of 176 of the 191 clinical specimens sequenced on the S5XL system and prepared using the SARS-CoV-2 research panel had nearly complete coverage (>98%) of the viral genome, with an average depth of 5,031×. Similar coverage levels (>98%) were observed for 81/83 primary specimens that were sequenced with both methods tested. The sample with the lowest viral load (CT value of 32.54) achieved 89% coverage using the MiSeq method and failed to sequence with the AmpliSeq method. Consensus sequences produced by each method were identical for 81/82 samples in areas of equal coverage, with a single difference present in one sample. The AmpliSeq approach is as effective as the Illumina-based method using ARTIC v3 amplification for sequencing SARS-CoV-2 directly from patient specimens across a range of viral loads (CT values of 15.56 to 32.54 [median, 22.18]). The AmpliSeq workflow is very easily automated with the Ion Chef and S5 instruments and requires less training and experience with next-generation sequencing sample preparation than the Illumina workflow.

Genomics, social media and mobile phone data enable mapping of SARS-CoV-2 lineages to inform health policy in Bangladesh.

Genomics, combined with population mobility data, used to map importation and spatial spread of SARS-CoV-2 in high-income countries has enabled the implementation of local control measures. Here, to track the spread of SARS-CoV-2 lineages in Bangladesh at the national level, we analysed outbreak trajectory and variant emergence using genomics, Facebook 'Data for Good' and data from three mobile phone operators. We sequenced the complete genomes of 67 SARS-CoV-2 samples (collected by the IEDCR in Bangladesh between March and July 2020) and combined these data with 324 publicly available Global Initiative on Sharing All Influenza Data (GISAID) SARS-CoV-2 genomes from Bangladesh at that time. We found that most (85%) of the sequenced isolates were Pango lineage B.1.1.25 (58%), B.1.1 (19%) or B.1.36 (8%) in early-mid 2020. Bayesian time-scaled phylogenetic analysis predicted that SARS-CoV-2 first emerged during mid-February in Bangladesh, from abroad, with the first case of coronavirus disease 2019 (COVID-19) reported on 8 March 2020. At the end of March 2020, three discrete lineages expanded and spread clonally across Bangladesh. The shifting pattern of viral diversity in Bangladesh, combined with the mobility data, revealed that the mass migration of people from cities to rural areas at the end of March, followed by frequent travel between Dhaka (the capital of Bangladesh) and the rest of the country, disseminated three dominant viral lineages. Further analysis of an additional 85 genomes (November 2020 to April 2021) found that importation of variant of concern Beta (B.1.351) had occurred and that Beta had become dominant in Dhaka. Our interpretation that population mobility out of Dhaka, and travel from urban hotspots to rural areas, disseminated lineages in Bangladesh in the first wave continues to inform government policies to control national case numbers by limiting within-country travel.

Occurrence and distribution of compound microsatellites in the genomes of three economically important virus families.

Microsatellites are nonrandom hypervariable iterations of one to six nucleotides, existing across the coding as well as noncoding regions of virtually all known genomes, arising primarily due to polymerase slippage and unequal crossing over during replication events. Two or more perfect microsatellites located in close proximity form compound microsatellites. We studied the distribution of compound microsatellites in 118 ssDNA virus genomes belonging to three economically important virus families, namely Anelloviridae, Circoviridae, and Parvoviridae, known to predominantly infect livestock and humans. Among these virus families, 0-58.49% of perfect microsatellites were involved in the formation of compound microsatellites, the majority being located in the coding regions. No clear relationship existed between the genomic features (genome size and GC%) and compound microsatellite characteristics (relative abundance and relative density). The majority of the compound microsatellites resulted from di-SSR couples. A strong positive relationship was observed between the maximum distance value and length of compound microsatellite, percentage of microsatellites involved in the compound microsatellite formation, and relative microsatellite density. The degree of variability among microsatellite characteristics studied was largely a species-specific phenomenon. A major proportion of compound microsatellites was represented by similar motif combinations. The findings of the present study will help in better understanding of the structural, functional, and evolutionary role of compound microsatellites prevailing in the smaller genomes.

The Inclusive Review on SARS-CoV-2 Biology, Epidemiology, Diagnosis, and Potential Management Options.

A novel coronavirus member was reported in Wuhan City, Hubei Province, China, at the end of the year 2019. Initially, the infection spread locally, affecting the Wuhan people, and then expanded rapidly throughout the world. On 11 March 2020, the World Health Organization (WHO) proclaimed it a global pandemic. The virus is a new strain most closely related to a bat coronavirus (RaTG13) which was not previously discovered in humans and is now formally known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) is the disease syndrome that the SARS-CoV-2 virus triggers. It is suggested that SARS-CoV-2 can be transmitted through aerosols, direct/indirect contact, and also during medical procedures and specimen handling. The infection is characterized by isolated flu-like symptoms, but there may be specific signs of fever, fatigue, cough, and shortness of breath, as well as the loss of smell and breathing difficulty. Within this report, we tried to review the most current scientific literature published by January 2021 on various aspects of the outbreak, including virus structure, pathogenesis, clinical presentation, epidemiology, diagnostic approaches, potential therapeutics and vaccines, and prospects. We hope this article makes a beneficial impact on public education to better deal with the SARS-CoV-2 crisis and push a step forward in the near term towards its prevention and control.

Preliminary assessment of viral metagenome from cancer tissue and blood from patients with lung adenocarcinoma.

In this study, using a viral metagenomic method, we investigated the composition of virome in blood and cancer tissue samples that were collected from 25 patients with lung adenocarcinoma. Results indicated that virus sequences showing similarity to human pegivirus (HPgV), anellovirus, human endogenous retrovirus (HERV), and polyomavirus were recovered from this cohort. Three different complete genomes of HPgV were acquired from the blood samples and one complete genome of polyomavirus was determined from the cancer tissue sample. Phylogenetic analysis indicated that the three HPgV strains belonged to genotype 3 and the polyomavirus showed the highest sequence identity (99.73%) to trichodysplasia spinulosa-associated polyomavirus. PCR screening results indicated that the three HPgVs were present in 5 out of the 25 blood samples and the polyomavirus only existed in a cancer tissue sample pool. Whether infections with viruses have an association with lung cancer needs further study with a larger size of sampling.

A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2.

BACKGROUND: The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. RESULTS: Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach. CONCLUSIONS: The tailed amplicon method we describe represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

Genetic diversity, haplotype analysis, and risk factor assessment of hepatitis A virus isolates from the West Bank, Palestine during the period between 2014 and 2016.

BACKGROUND: Hepatitis A virus (HAV) infection is one of the major causes of acute viral hepatitis. HAV genotypes and its genetic diversity is rarely investigated in our region as well as worldwide. AIMS: The aims of the present study were to determine the HAV genotypes and its risk factors and to investigate the genetic diversity of the HAV isolates in the West Bank, Palestine. STUDY DESIGN: A cohort of 161 clinically and laboratory-confirmed HAV (IgM-positive) cases and 170 apparently healthy controls from all the districts of the West Bank, Palestine during the period of 2014 to 2016 were tested for HAV infection using IgM antibodies, RT-PCR and sequence analysis of the VP3/VP1 junction region of the HAV genome. Phylogenetic analysis, genetic diversity and haplotypes analysis were used to characterize the VP3/VP1 sequences. RESULTS: All the 34 sequences of the HAV were found to be of HAV-IB sub-genotype. The phylogenetic analysis showed four main clusters with cluster III exclusively consisting of 18 Palestinian isolates (18/23-78%), but with weak bootstrap values. A high haplotype diversity (Hd) and low nucleotide diversity (π) were observed. Cluster III showed high number of haplotypes (h = 8), but low haplotype (gene) diversity (Hd = 0.69). A total of 28 active haplotypes with some consisting of more than one sequence were observed using haplotype network analysis. The Palestinian haplotypes are characterized by closely related viral haplotypes with one SNV away from each other which ran parallel to cluster III in the phylogenetic tree. A smaller Palestinian haplotype (4 isolates) was three SNVs away from the major haplotype cluster (n = 10) and closer to others haplotypes from Iran, Spain, and South Africa. Young age, low level of parent's education, infrequent hand washing before meals, and drinking of un-treated water were considered the major HAV risk factors in the present study. CONCLUSION: Haplotype network analysis revealed haplotype variation among the HAV Palestinian sequences despite low genetic variation and nucleotide diversity. In addition, this study reconfirmed that age and parent's level of education as HAV risk factors, while hand washing and treating drinking water as protective factors.

SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification.

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.

Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19.

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.