VirusTaxo: Taxonomic classification of viruses from the genome sequence using k-mer enrichment

Classification of viruses into their taxonomic ranks (e.g., order, family, and genus) provides a framework to organize an abundant population of viruses. Next-generation metagenomic sequencing technologies lead to a rapid increase in generating sequencing data of viruses which require bioinformatics tools to analyze the taxonomy. Many metagenomic taxonomy classifiers have been developed to study microbiomes, but it is particularly challenging to assign the taxonomy of diverse virus sequences and there is a growing need for dedicated methods to be developed that are optimized to classify virus sequences into their taxa. For taxonomic classification of viruses from metagenomic sequences, we developed VirusTaxo using diverse (e.g., 402 DNA and 280 RNA) genera of viruses. VirusTaxo has an average accuracy of 93% at genus level prediction in DNA and RNA viruses. VirusTaxo outperformed existing taxonomic classifiers of viruses where it assigned taxonomy of a larger fraction of metagenomic contigs compared to other methods. Benchmarking of VirusTaxo on a collection of SARS-CoV-2 sequencing libraries and metavirome datasets suggests that VirusTaxo can characterize virus taxonomy from highly diverse contigs and provide a reliable decision on the taxonomy of viruses.     

Metagenomic Profile of the Viral Communities in Rhipicephalus spp. Ticks from Yunnan,China

Besides mosquitoes, ticks are regarded as the primary source of vector-borne infectious diseases. Indeed, a wide variety of severe infectious human diseases, including those involving viruses, are transmitted by ticks in many parts of the world. To date, there are no published reports on the use of next-generation sequencing for studying viral diversity in ticks or discovering new viruses in these arthropods from China. Here, Ion-torrent sequencing was used to investigate the presence of viruses in three Rhipicephalus spp. tick pools (NY-11, NY-13, and MM-13) collected from the Menglian district of Yunnan, China. The sequencing run resulted in 3,641,088, 3,106,733, and 3,871,851 reads in each tick pool after trimming. Reads and assembled contiguous sequences (contigs) were subject to basic local alignment search tool analysis against the GenBank database. Large numbers of reads and contigs related to known viral sequences corresponding to a broad range of viral families were identified. Some of the sequences originated from viruses that have not been described previously in ticks. Our findings will facilitate better understanding of the tick virome, and add to our current knowledge of disease-causing viruses in ticks living under natural conditions.     

Molecular epidemiology of parvoviruses

Molecular epidemiological studies of paruoviruses are complicated by the low levels of variation in the DNA genome of the viruses. Nevertheless, use of restriction enzyme analysis, DNA sequencing and monoclonal antibody analysis of epitope variation has been used to examine the distribution of virus strains in nature. Comparison of canine parvovirus and the related viruses from other carnivores showed that a number of variant strains of the viruses could be identfied by DNA sequence and antigenic analysis. Among CPV isolates some strains replaced previous antigenic types, while other variant antigenic types coexisted in nature. The viruses are apparently able to spread world wide, as most geographically separated viruses are not genetically distinct. The human B19 parvovirus has been examined by restriction enzyme analysis and limited DNA sequencing, and a variety of characteristic strains identified. Some were found world wide, while others were apparently found to be associated with certain countries.     

Metagenomic analysis of human diarrhea: viral detection and discovery

Worldwide, approximately 1.8 million children die from diarrhea annually, and millions more suffer multiple episodes of nonfatal diarrhea. On average, in up to 40% of cases, no etiologic agent can be identified. The advent of metagenomic sequencing has enabled systematic and unbiased characterization of microbial populations; thus, metagenomic approaches have the potential to define the spectrum of viruses, including novel viruses, present in stool during episodes of acute diarrhea. The detection of novel or unexpected viruses would then enable investigations to assess whether these agents play a causal role in human diarrhea. In this study, we characterized the eukaryotic viral communities present in diarrhea specimens from 12 children by employing a strategy of "micro-mass sequencing" that entails minimal starting sample quantity (<100 mg stool), minimal sample purification, and limited sequencing (384 reads per sample). Using this methodology we detected known enteric viruses as well as multiple sequences from putatively novel viruses with only limited sequence similarity to viruses in GenBank.     

Mining livestock genome datasets for an unconventional characterization of animal DNA viromes

Whole genome sequencing (WGS) datasets, usually generated for the investigation of the individual animal genome, can be used for additional mining of the fraction of sequencing reads that remains unmapped to the respective reference genome. A significant proportion of these reads contains viral DNA derived from viruses that infected the sequenced animals. In this study, we mined more than 480 billion sequencing reads derived from 1471 WGS datasets produced from cattle, pigs, chickens and rabbits. We identified 367 different viruses among which 14, 11, 12 and 1 might specifically infect the cattle, pig, chicken and rabbit, respectively. Some of them are ubiquitous, avirulent, highly or potentially damaging for both livestock and humans. Retrieved viral DNA information provided a first unconventional and opportunistic landscape of the livestock viromes that could be useful to understand the distribution of some viruses with potential deleterious impacts on the animal food production systems.     

Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies

Current knowledge of plant virus diversity is biased towards agents of visible and economically important diseases. Less is known about viruses that have not caused major diseases in crops, or viruses from native vegetation, which are a reservoir of biodiversity that can contribute to viral emergence. Discovery of these plant viruses is hindered by the traditional approach of sampling individual symptomatic plants. Since many damaging plant viruses are transmitted by insect vectors, we have developed "vector-enabled metagenomics" (VEM) to investigate the diversity of plant viruses. VEM involves sampling of insect vectors (in this case, whiteflies) from plants, followed by purification of viral particles and metagenomic sequencing. The VEM approach exploits the natural ability of highly mobile adult whiteflies to integrate viruses from many plants over time and space, and leverages the capability of metagenomics for discovering novel viruses. This study utilized VEM to describe the DNA viral community from whiteflies (Bemisia tabaci) collected from two important agricultural regions in Florida, USA. VEM successfully characterized the active and abundant viruses that produce disease symptoms in crops, as well as the less abundant viruses infecting adjacent native vegetation. PCR assays designed from the metagenomic sequences enabled the complete sequencing of four novel begomovirus genome components, as well as the first discovery of plant virus satellites in North America. One of the novel begomoviruses was subsequently identified in symptomatic Chenopodium ambrosiodes from the same field site, validating VEM as an effective method for proactive monitoring of plant viruses without a priori knowledge of the pathogens. This study demonstrates the power of VEM for describing the circulating viral community in a given region, which will enhance our understanding of plant viral diversity, and facilitate emerging plant virus surveillance and management of viral diseases.     

Autologous Antibody Capture to Enrich Immunogenic Viruses for Viral Discovery

Discovery of new viruses has been boosted by novel deep sequencing technologies. Currently, many viruses can be identified by sequencing without knowledge of the pathogenicity of the virus. However, attributing the presence of a virus in patient material to a disease in the patient can be a challenge. One approach to meet this challenge is identification of viral sequences based on enrichment by autologous patient antibody capture. This method facilitates identification of viruses that have provoked an immune response within the patient and may increase the sensitivity of the current virus discovery techniques. To demonstrate the utility of this method, virus discovery deep sequencing (VIDISCA-454) was performed on clinical samples from 19 patients: 13 with a known respiratory viral infection and 6 with a known gastrointestinal viral infection. Patient sera was collected from one to several months after the acute infection phase. Input and antibody capture material was sequenced and enrichment was assessed. In 18 of the 19 patients, viral reads from immunogenic viruses were enriched by antibody capture (ranging between 1.5x to 343x in respiratory material, and 1.4x to 53x in stool). Enriched reads were also determined in an identity independent manner by using a novel algorithm Xcompare. In 16 of the 19 patients, 21% to 100% of the enriched reads were derived from infecting viruses. In conclusion, the technique provides a novel approach to specifically identify immunogenic viral sequences among the bulk of sequences which are usually encountered during virus discovery metagenomics.     

Virus identification in unknown tropical febrile illness cases using deep sequencing

Dengue virus is an emerging infectious agent that infects an estimated 50-100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic pathogen in ～40% of dengue-like illnesses. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non-diagnosable disease challenges. In this study, we used the Virochip microarray and deep sequencing to characterize the spectrum of viruses present in human sera from 123 Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously deep sequence multiple serum specimens, generating on average over 1 million reads per sample. We then implemented a stepwise bioinformatic filtering pipeline to remove the majority of human and low-quality sequences to improve the speed and accuracy of subsequent unbiased database searches. By deep sequencing, we were able to detect virus sequence in 37% (45/123) of previously negative cases. These included 13 cases with Human Herpesvirus 6 sequences. Other samples contained sequences with similarity to sequences from viruses in the Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In some cases, the putative viral sequences were virtually identical to known viruses, and in others they diverged, suggesting that they may derive from novel viruses. These results demonstrate the utility of unbiased metagenomic approaches in the detection of known and divergent viruses in the study of tropical febrile illness.     

Is There Still Room for Novel Viral Pathogens in Pediatric Respiratory Tract Infections?

Viruses are the most frequent cause of respiratory disease in children. However, despite the advanced diagnostic methods currently in use, in 20 to 50% of respiratory samples a specific pathogen cannot be detected. In this work, we used a metagenomic approach and deep sequencing to examine respiratory samples from children with lower and upper respiratory tract infections that had been previously found negative for 6 bacteria and 15 respiratory viruses by PCR. Nasal washings from 25 children (out of 250) hospitalized with a diagnosis of pneumonia and nasopharyngeal swabs from 46 outpatient children (out of 526) were studied. DNA reads for at least one virus commonly associated to respiratory infections was found in 20 of 25 hospitalized patients, while reads for pathogenic respiratory bacteria were detected in the remaining 5 children. For outpatients, all the samples were pooled into 25 DNA libraries for sequencing. In this case, in 22 of the 25 sequenced libraries at least one respiratory virus was identified, while in all other, but one, pathogenic bacteria were detected. In both patient groups reads for respiratory syncytial virus, coronavirus-OC43, and rhinovirus were identified. In addition, viruses less frequently associated to respiratory infections were also found. Saffold virus was detected in outpatient but not in hospitalized children. Anellovirus, rotavirus, and astrovirus, as well as several animal and plant viruses were detected in both groups. No novel viruses were identified. Adding up the deep sequencing results to the PCR data, 79.2% of 250 hospitalized and 76.6% of 526 ambulatory patients were positive for viruses, and all other children, but one, had pathogenic respiratory bacteria identified. These results suggest that at least in the type of populations studied and with the sampling methods used the odds of finding novel, clinically relevant viruses, in pediatric respiratory infections are low.     

Simultaneous virus identification and characterization of severe unexplained pneumonia cases using a metagenomics sequencing technique

Many viruses can cause respiratory diseases in humans. Although great advances have been achieved in methods of diagnosis, it remains challenging to identify pathogens in unexplained pneumonia (UP) cases. In this study, we applied next-generation sequencing (NGS) technology and a metagenomic approach to detect and characterize respiratory viruses in UP cases from Guizhou Province, China. A total of 33 oropharyngeal swabs were obtained from hospitalized UP patients and subjected to NGS. An unbiased metagenomic analysis pipeline identified 13 virus species in 16 samples. Human rhinovirus C was the virus most frequently detected and was identified in seven samples. Human measles virus, adenovirus B 55 and coxsackievirus A10 were also identified. Metagenomic sequencing also provided virus genomic sequences, which enabled genotype characterization and phylogenetic analysis. For cases of multiple infection, metagenomic sequencing afforded information regarding the quantity of each virus in the sample, which could be used to evaluate each viruses’ role in the disease. Our study highlights the potential of metagenomic sequencing for pathogen identification in UP cases.     

A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.     

中国部分地区蝙蝠携带病毒的宏基因组学分析

蝙蝠携带有60多种病毒,其中许多对人有高度致病性.为了解中国蝙蝠携带病毒的自然本底、蝙蝠病毒的多样性和挖掘潜在的病毒病原,通过基于Solexa高通量测序的病毒宏基因组学技术对从吉林、云南、湖南采集的蝙蝠组织进行病毒组学研究,获得了11 644 232条读长(Reads),并拼接出44 872条重叠序列(Contig).通过核酸序列注释发现,其中8.2％(4 002/44 872)的重叠序列与病毒相关,能进一步注释到36个病毒科,包括19种脊椎动物病毒、6种植物病毒、4种昆虫病毒和4种噬菌体.通过对重叠序列的遗传进化分析、多序列比对显示,被注释为细小病毒、腺联病毒、博卡病毒、腺病毒、小双节RNA病毒等的重叠序列与已知病毒相似,部分序列却又呈现出明显的序列差异.通过对腺病毒和博卡病毒进一步的PCR扩增证实了此研究方法可靠.旨在了解我国蝙蝠携带病毒组的构成,对建立高效的野生动物源人兽共患病的监测方法提供参考.     

Identification of Novel Viruses Using VirusHunter -- an Automated Data Analysis Pipeline

Quick and accurate identification of microbial pathogens is essential for both diagnosis and response to emerging infectious diseases. The advent of next-generation sequencing technology offers an unprecedented platform for rapid sequencing-based identification of novel viruses. We have developed a customized bioinformatics data analysis pipeline, VirusHunter, for the analysis of Roche/454 and other long read Next generation sequencing platform data. To illustrate the utility of VirusHunter, we performed Roche/454 GS FLX titanium sequencing on two unclassified virus isolates from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). VirusHunter identified sequences derived from a novel bunyavirus and a novel reovirus in the two samples respectively. Further sequence analysis demonstrated that the viruses were novel members of the Phlebovirus and Orbivirus genera. Both Phlebovirus and Orbivirus genera include many economic important viruses or serious human pathogens.     

浙江地区鼬獾和犬源狂犬病病毒分离株全基因组测序与分析

测定浙江地区狂犬病病毒分离株(鼬獾和犬)全基因组序列,从分子水平对病毒进行遗传变异特征分析,了解狂犬病病毒在浙江的流行和变异情况以及目前浙江流行株的遗传学背景,以丰富中国狂犬病病毒街毒流行株的全基因组信息。脑内接种1~2日乳鼠分离狂犬病病毒,RT-PCR反应测定浙江地区狂犬病病毒分离株全基因组核苷酸序列,并进行编码蛋白和序列相似性比较及种系发生分析。测序获得狂犬病病毒浙江淳安鼬獾分离株F02、F04和松阳犬分离株D01、D02全基因组核苷酸序列信息:基因组全长11 923和11 925 nts,前导序列Leader长58nts,5个ORF为:NP(1 353 nts);PP(894 nts);MP(609 nts);GP(1 575 nts);LP(6 386 nts),N-P-M-G间隔序列长2、5、5 nts;G-L基因间的伪基因Ψ长423 nts;Trailer尾长70 nts。核酸BLAST及多重序列比对分析显示浙江地区4个狂犬病病毒分离株的全基因组序列的组成和结构符合弹状病毒科狂犬病病毒属的特征;中国毒株之间特别是浙江同种动物狂犬病病毒之间各个基因区域核苷酸与氨基酸序列相似性最高,浙江病毒全基因组序列编码蛋白氨基酸序列相似性高于核苷酸序列相似性,说明蛋白质编码基因的核苷酸变异大多属于同义突变;浙江病毒负链RNA基因组5个基因编码氨基酸的长度没有变异,5个编码蛋白仅表现较少的序列变化;浙江病毒与本研究选择的代表性引用街毒株或者来自街毒的减毒株的变异位点和变异类型相似,多重序列相似性的比较和种系发生分析显示所分离的狂犬病病毒浙江街毒株均属于基因1型,具有较独特的中国地域性特点,故本研究中的浙江地区分离株极有可能是自然界中固有的街毒株。     

Typing of pestiviruses from eland in Zimbabwe

Pestiviruses were isolated from three eland (Taurotragus oryx) in Zimbabwe. The viruses were characterised by typing with monoclonal antibodies and by partial genetic sequencing. All were similar to bovine viral diarrhea viruses commonly isolated from cattle. This suggests that bovine viral diarrhea virus can spread from cattle to eland.     

From deep sequencing to viral tagging: Recent advances in viral metagenomics

Culture‐independent high‐throughput sequencing has provided unprecedented insights into microbial ecology, particularly for Earth's most ubiquitous and diverse inhabitants – the viruses. A plethora of methods now exist for amplifying the vanishingly small amounts of nucleic acids in natural viral communities in order to sequence them, and sequencing depth is now so great that viral genomes can be detected and assembled even amid large concentrations of non‐viral DNA. Complementing these advances in amplification and sequencing is the ability to physically link fluorescently labeled viruses to their host cells via high‐throughput flow sorting. Sequencing of such isolated virus–host pairs facilitates cultivation‐independent exploration of the natural host range of viruses. Within the next decade, as these technologies become widespread, we can expect to see a systematic expansion of our knowledge of viruses and their hosts.     

Recent advances and prospects in Prunus virology

The stone fruit genus Prunus, within the family Rosaceae, comprises more than 230 species, some of which have great importance or value as ornamental or fruit crops. Prunus are affected by numerous viruses and viroids linked to the vegetative propagation practices in many of the cultivated species. To date, 44 viruses and three viroids have been described in the 9 main cultivated Prunus species. Seven of these viruses and one viroid have been identified in Prunus hosts within the last 5 years. This work addresses recent advances and prospects in the study of viruses and viroids affecting Prunus species, mostly concerning the detection and characterisation of the agents involved, pathogenesis analysis and the search for new control tools. New sequencing technologies are quickly reshaping the way we can identify and characterise new plant viruses and isolates. Specific efforts aimed at virus identification or data mining of high‐throughput sequencing data generated for plant genomics‐oriented purposes can efficiently reveal the presence of known or novel viruses. These technologies have also been used to gain a deeper knowledge of the pathogenesis mechanisms at the gene and miRNA expression level that underlie the interactions between Prunus spp. and their main viruses and viroids. New biotechnological control tools include the transfer of resistance by grafting, the use of new sources of resistance and the development of gene silencing strategies using genetic transformation. In addition, the application of next generation sequencing and genome editing techniques will contribute to improving our knowledge of virus–host interactions and the mechanisms of resistance. This should be of great interest in the search to obtain new Prunus cultivars capable of dealing both with known viruses and viroids and with those that are yet to be discovered in the uncertain scenario of climate change.     

Identification of Multiple Novel Viruses,Including a Parvovirus and a Hepevirus,in Feces of Red Foxes

Red foxes (Vulpes vulpes) are the most widespread members of the order of Carnivora. Since they often live in (peri)urban areas, they are a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. Here we evaluated the fecal viral microbiome of 13 red foxes by random PCR in combination with next-generation sequencing. Various novel viruses, including a parvovirus, bocavirus, adeno-associated virus, hepevirus, astroviruses, and picobirnaviruses, were identified.     

New DNA viruses identified in patients with acute viral infection syndrome

A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.