Complete Viral Genome Research Articles

Evaluation of un-methylated DNA enrichment in sequencing of African swine fever virus complete genome

African swine fever is a febrile hemorrhagic fever disease that is caused by the African swine fever virus (ASFV) and is lethal for domestic pigs and wild boar. ASFV also infects soft ticks of the genus Ornithodoros, some species of which can act as a vector for ASFV. Whole genome sequencing of ASFV is a challenge because, due to the size difference of the host genome versus the viral genome, the higher proportion of host versus virus DNA fragments renders the virus sequencing poorly efficient. A novel approach of DNA enrichment, based on the separation of methylated and un-methylated DNA, has been reported but without an evaluation of its efficacy. In this study, the efficiency of the un-methylated DNA enrichment protocol was evaluated for pig and tick samples infected by ASFV. As expected, fewer reads corresponding to ASFV were found in the methylated fraction compared to the un-methylated fraction. However, the sequencing coverage of the un-methylated fraction was not improved compared to the untreated DNA. In our hands, the ASFV DNA enrichment was inefficient for tick samples and very limited for pig samples. This enrichment process represents extra work and cost without a significant improvement of ASFV genome coverage. The efficiency of this enrichment approach and the cost/benefit ratio are discussed.

Building the National SARS-CoV-2 Laboratory Diagnostic Capacity in Taiwan.

Through early and proactive laboratory testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes novel coronavirus 2019, Taiwan has demonstrated an efficient and rapid control response to contain the outbreak. Two days after the World Health Organization announced the complete viral genome sequence, the national laboratory of the Taiwan Centers for Disease Control developed a specific real-time reverse transcription polymerase chain reaction (real-time RT-PCR) test for SARS-CoV-2. The national laboratory network was further strengthened through the recruitment of medical centers and regional hospitals distributed throughout most geographical regions of the country. Ultimately, a network of 60 laboratories with a capacity of 7,342 real-time RT-PCR tests per day was established. Between January 14 and August 5, 2020, a total of 158,772 tests were conducted, corresponding to 120,487 cases. Test results were obtained within 24 hours, enabling an efficient and rapid control response.

Tobamoviruses can be frequently present in the oropharynx and gut of infants during their first year of life

Plant viruses have been reported to be common in the gut of human adults, presumably as result of food ingestion. In this work, we report that plant viruses can also be found frequently in the gut and oropharynx of children during their first year of life, even when they are exclusively breast-fed. Fecal and oropharynx samples were collected monthly, from birth to 1 year of age, from three apparently healthy children in a semi-rural community and analyzed by next generation sequencing. In 100% of the fecal samples and 65% of the oropharynx samples at least one plant virus was identified. Tobamoviruses in the Virgaviridae family were by far the most frequently detected, with tropical soda apple mosaic virus, pepper mild mottle virus, and opuntia tobamovirus 2 being the most common species. Seventeen complete virus genomes could be assembled, and phylogenetic analyses showed a large diversity of virus strains circulating in the population. These results suggest that children are continuously exposed to an extensive and highly diverse collection of tobamoviruses. Whether the common presence of plant viruses at an early age influences the infant’s immune system, either directly or through interaction with other members of the microbiota, remains to be investigated.

Viral diversity in oral cavity from Sapajus nigritus by metagenomic analyses.

Sapajus nigritus are non-human primates which are widespread in South America. They are omnivores and live in troops of up to 40 individuals. The oral cavity is one of the main entry routes for microorganisms, including viruses. Our study proposed the identification of viral sequences from oral swabs collected in a group of capuchin monkeys (n= 5) living in a public park in a fragment of Mata Atlantica in South Brazil. Samples were submitted to nucleic acid extraction and enrichment, which was followed by the construction of libraries. After high-throughput sequencing and contig assembly, we used a pipeline to identify 11 viral families, which are Herpesviridae, Parvoviridae, Papillomaviridae, Polyomaviridae, Caulimoviridae, Iridoviridae, Astroviridae, Poxviridae, and Baculoviridae, in addition to two complete viral genomes of Anelloviridae and Genomoviridae. Some of these viruses were closely related to known viruses, while other fragments are more distantly related, with 50% of identity or less to the currently available virus sequences in databases. In addition to host-related viruses, insect and small vertebrate-related viruses were also found, as well as plant-related viruses, bringing insights about their diet. In conclusion, this viral metagenomic analysis reveals, for the first time, the profile of viruses in the oral cavity of wild, free ranging capuchin monkeys.

Genome reconstruction of white spot syndrome virus (WSSV) from archival Davidson\u2019s-fixed paraffin embedded shrimp (Penaeus vannamei) tissue

Formalin-fixed paraffin-embedded (FFPE) tissues are a priceless resource for diagnostic laboratories worldwide. However, DNA extracted from these tissues is often not optimal for most downstream molecular analysis due to fragmentation and chemical modification. In this study, the complete genome of white spot syndrome virus (WSSV) was reconstructed from ~ 2-year-old archived Davidson’s-fixed paraffin-embedded (DFPE) shrimp tissue using Next Generation Sequencing (NGS). A histological analysis was performed on archived DFPE shrimp tissue and a sample showing a high level of WSSV infection was selected for molecular analysis. The viral infection was further confirmed by molecular methods. DNA isolated from DFPE and fresh frozen (FF) tissues were sequenced by NGS. The complete genome reconstruction of WSSV (~ 305 kbp) was achieved from both DFPE and FF tissue. Single nucleotide polymorphisms, insertion and deletions were compared between the genomes. Thirty-eight mutations were identified in the WSSV genomes from the DFPE and FF that differed from the reference genome. This is the first study that has successfully sequenced the complete genome of a virus of over 300 kbp from archival DFPE tissue. These findings demonstrate that DFPE shrimp tissue represents an invaluable resource for prospective and retrospective studies, evolutionary studies and opens avenues for pathogen discovery.

Extraordinary diversity of viruses in deep-sea sediments as revealed by metagenomics without prior virion separation.

Our current knowledge of the virosphere in deep-sea sediments remains rudimentary. Here we investigated viral diversity at both gene and genomic levels in deep-sea sediments of Southwest Indian Ocean. Analysis of 19 676 106 non-redundant genes from the metagenomic DNA sequences revealed a large number of unclassified viral groups in these samples. A total of 1106 high-confidence viral contigs were obtained after two runs of assemblies, and 217 of these contigs with sizes up to ~120 kb were shown to represent complete viral genomes. These contigs are clustered with no known viral genomes, and over 2/3 of the ORFs on the viral contigs encode no known functions. Furthermore, most of the complete viral contigs show limited similarity to known viral genomes in genome organization. Most of the classified viral contigs are derived from dsDNA viruses belonging to the order Caudovirales, including primarily members of the families Myoviridae, Podoviridae and Siphoviridae. Most of these viruses infect Proteobacteria and, less frequently, Planctomycetes, Firmicutes, Chloroflexi, etc. Auxiliary metabolic genes (AMGs), present in abundance on the viral contigs, appear to function in modulating the host ability to sense environmental gradients and community changes, and to uptake and metabolize nutrients.

Simultaneous Viral Whole-Genome Sequencing and Differential Expression Profiling in Respiratory Syncytial Virus Infection of Infants.

Targeted metagenomics using strand-specific libraries with target enrichment is a sensitive, generalized approach to pathogen sequencing and transcriptome profiling. Using this method, we recovered 13 (76%) complete human respiratory syncytial virus (RSV) genomes from 17 clinical respiratory samples, reconstructed the phylogeny of the infecting viruses, and detected differential gene expression between 2 RSV subgroups, specifically, a lower expression of the P gene and a higher expression of the M2 gene in RSV-A than in RSV-B. This methodology can help to relate viral genetics to clinical phenotype and facilitate ongoing population-level RSV surveillance and vaccine development. Clinical Trials Registration. NCT03627572 and NCT03756766.

High-Quality Resolution of the Outbreak-Related Zika Virus Genome and Discovery of New Viruses Using Ion Torrent-Based Metatranscriptomics.

Arboviruses, including the Zika virus, have recently emerged as one of the most important threats to human health. The use of metagenomics-based approaches has already proven valuable to aid surveillance of arboviral infections, and the ability to reconstruct complete viral genomes from metatranscriptomics data is key to the development of new control strategies for these diseases. Herein, we used RNA-based metatranscriptomics associated with Ion Torrent deep sequencing to allow for the high-quality reconstitution of an outbreak-related Zika virus (ZIKV) genome (10,739 nt), with extended 5′-UTR and 3′-UTR regions, using a newly-implemented bioinformatics approach. Besides allowing for the assembly of one of the largest complete ZIKV genomes to date, our strategy also yielded high-quality complete genomes of two arthropod-infecting viruses co-infecting C6/36 cell lines, namely: Alphamesonivirus 1 strain Salvador (20,194 nt) and Aedes albopictus totivirus-like (4618 nt); the latter likely represents a new viral species. Altogether, our results demonstrate that our bioinformatics approach associated with Ion Torrent sequencing allows for the high-quality reconstruction of known and unknown viral genomes, overcoming the main limitation of RNA deep sequencing for virus identification.

Blueberry red ringspot virus genomes from Florida inferred through analysis of blueberry root transcriptomes

A growing number of metagenomics-based approaches have been used for the discovery of viruses in insects, cultivated plants, and water in agricultural production systems. In this study, sixteen blueberry root transcriptomes from eight clonally propagated blueberry plants of cultivar ‘Emerald’ (interspecific hybrid of Vaccinium corymbosum and V. darrowi) generated as part of a separate study on varietal tolerance to soil salinity were analyzed for plant viral sequences. The objective was to determine if the asymptomatic plants harbored the latent blueberry red ringspot virus (BRRV) in their roots. The only currently known mechanism of transmission of BRRV is through vegetative propagation; however, the virus can remain latent for years with some plants of ‘Emerald’ never developing red ringspot symptoms. Bioinformatic analyses of ‘Emerald’ transcriptomes using de novo assembly and reference-based mapping approaches yielded eight complete viral genomes of BRRV (genus Soymovirus, family Caulimoviridae). Validation in vitro by PCR confirmed the presence of BRRV in 100% of the ‘Emerald’ root samples. Sequence and phylogenetic analyses showed 94% to 97% nucleotide identity between BRRV genomes from Florida and sequences from Czech Republic, Japan, Poland, Slovenia, and the United States. Taken together, this study documented the first detection of a complete BRRV genome from roots of asymptomatic blueberry plants and in Florida through in silico analysis of plant transcriptomes.

Genetic variants and source of introduction of SARS-CoV-2 in South America.

After more than 4 months of the COVID‐19 pandemics with genomic information of SARS‐CoV‐2 around the globe, there are more than 1000 complete genomes of this virus. We used 691 genomes from the GISAID database. Several studies have been reporting mutations and hotspots according to viral evolution. Our work intends to show and compare positions that have variants in 30 complete viral genomes from South American countries. We classified strains according to point alterations and portray the source where strains came into this region. Most viruses entered South America from Europe, followed by Oceania. Only Chilean isolates demonstrated a relationship with Asian isolates. Some changes in South American genomes are near to specific domains related to viral replication or the S protein. Our work contributes to the global understanding of which sort of strains are spreading throughout South America, and the differences among them according to the first isolates introduced to this region.

Equine pegiviruses cause persistent infection of bone marrow and are not associated with hepatitis.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1–4% of healthy individuals and another 5–13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler’s disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler’s disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3’ terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2–3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.

An ancient view on host pathogen interaction across time and space.

The ancient DNA revolution provided diverse fields with an unprecedented opportunity to look back into the past and shed light on research aspects that were until now subject to speculation and inference from modern data. In particular enrichment methods that allow the targeted retrieval of millions of SNP positions from ancient human genomes, or even complete bacterial and viral genomes have the potential to revolutionize our understanding of host pathogen interactions. Ancient DNA combined with new bioinformatic tools now even allows actual allele calling for immunogenetic systems such as Human Leukocyte Antigen (HLA) across time and space. The coming years will provide us with frequency data of human immunity genes, such as HLA, as well as genome wide data of ancient pathogens from many time periods of human history, and will therefore provide us with a dynamic view on historical human adaptation to pathogen exposure on a population wide scale.

Dhati Welel virus, the missing mammarenavirus of the widespread Mastomys natalensis

The Natal multimammate mouse, Mastomys natalensis, occurs throughout sub-Saharan Africa. Mitochondrial phylogenetics indicate this species was fragmented during the Pleistocene, forming six matrilineage phylogroups: A-I, A-II, A-III, B-IV, B-V, B-VI with distinct ranges. All except the A-III lineage are identified as natural reservoirs of mammarenaviruses. M. natalensis A-III is found in western Ethiopia and is the only lineage reported in the country. While screening 203 small mammal samples from Dhati Welel National Park for mammarenaviruses, we detected mammarenavirus RNA in nine samples, eight from M. natalensis and one from M. awashensis. A sequence similarity search and phylogenetic analysis confirmed the M. natalensis mitochondrial DNA belongs to the A-III lineage. We characterised the complete virus genome, which showed typical mammarenavirus organisation. Phylogenetic analysis indicated it clusters with Gairo virus found in M. natalensis B-IV in Tanzania, while showing sufficient divergence from other mammarenaviruses to be considered as a new species, for which we proposed the name Dhati Welel. Additional sampling in the M. natalensis A-III phylogeographic range should help determine whether the detection of the virus in M. awashensis represents a local spill-over or if the virus circulates in both Mastomys species.

Camellia ringspot-associated virus 4, a proposed new foveavirus from Camellia japonica.

One large contig with high sequence similarity to Asian prunus virus 2 was identified by high-throughput sequencing from a camellia (Camellia japonica) tree with ringspot symptoms. The complete genome of this new virus was determined to be 8829 nucleotides long, excluding the 3' poly(A) tail. Its genome organization resembles that of known foveaviruses but contains an additional open reading frame in the 3'-terminal region. Phylogenetic analysis also places this virus with members of the genus Foveavirus in the family Betaflexiviridae in the same subgroup. The virus, which is provisionally named "camellia ringspot-associated virus 4″, shares 50-56% nucleotide sequence identity with other foveaviruses and should represent a new species in the genus.

A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein

SummaryThe unprecedented pandemic of pneumonia caused by a novel coronavirus, SARS-CoV-2, in China and beyond has had major public health impacts on a global scale [1, 2]. Although bats are regarded as the most likely natural hosts for SARS-CoV-2 [3], the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomic analysis of samples from 227 bats collected from Yunnan Province in China between May and October 2019. Notably, RmYN02 shares 93.3% nucleotide identity with SARS-CoV-2 at the scale of the complete virus genome and 97.2% identity in the 1ab gene, in which it is the closest relative of SARS-CoV-2 reported to date. In contrast, RmYN02 showed low sequence identity (61.3%) to SARS-CoV-2 in the receptor-binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, and in a similar manner to SARS-CoV-2, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the spike (S) protein. This provides strong evidence that such insertion events can occur naturally in animal betacoronaviruses.

Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

This protocol describes the production of human papillomavirus (HPV)-derived quasiviruses. Quasiviruses are infectious particles that are produced in 293TT packaging cells and contain a complete viral genome. We describe methods for infection of primary human keratinocytes with HPV quasiviruses, as well as assays to measure early viral DNA replication and transcription. Published 2020. U.S. Government. Basic Protocol 1: Transfection, harvest, and isolation of HPV quasiviruses Alternate Protocol 1: Packaging HPV DNA replicated in 293TT cells Alternate Protocol 2: Production of higher-purity quasivirus using the "Ripcord" method Support Protocol 1: Production of HPV minicircles Support Protocol 2: Production of recircularized HPV genomes Support Protocol 3: Screening of fractions for viral proteins Support Protocol 4: Screening of fractions for viral DNA Support Protocol 5: Measuring viral titer Support Protocol 6: Quantitation of quasivirions Basic Protocol 2: Infection of primary human foreskin keratinocytes with quasivirus Basic Protocol 3: HPV quasivirus transcription assay Basic Protocol 4: HPV quasivirus replication assay.

Characterization of the Full-Length Genome Sequences and Phylogenetic Analysis of HDV Strains Isolated from Patients with Chronic HBV and HDV Infection in Kyrgyz Republic

Objective. The purpose of our work was molecular genetic characterization of the hepatitis D virus isolates, circulating in the region with high prevalence of HBV + HDV super-infection. Materials and methods. The study material was 64 blood serum samples obtained from Kyrgyz Republic residents - patients with chronic viral hepatitis B+D. The hepatitis D virus complete genomes were sequenced, followed by phylogenetic analysis. Results and discussion. Based on the phylogenetic analysis of 64 HDV samples, it was shown that HDV genotype 1 (96.9 %) predominates in the examined group compared with HDV genotype 2 (3.1 %). Sequences were submitted to GenBank under access No MN984407 through MN984470. When assessing the genetic variability over the examined HDV genotype 1 samples, the maximum genetic distance was 12,49 %, and the minimum – 7,41 %. Within individual clusters, the genetic distance averaged from 2.6 % to 8.5 %. Among the sequences in GenBank, the closest resemblance to the HDV-2 Kyr41 and Kyr43 samples (nucleotide identity was 92.31 % and 89.57 %, respectively) was shown for the virus described earlier in Yakutia (AJ309880). To study the genetic relationships between the analyzed HDV genotype 1 strains in comparison with the HDV reference sequences, the predicted amino acid sequence was studied (111–214). Although hepatitis B preventive measures, including vaccination, have reduced the hepatitis D incidence, there is no effective way to prevent HDV infection in HBV carriers in endemic areas. The HDV sequence molecular-genetic characterization in this study, as well as the viral genomic sequence phylogenetic analysis, will help identify pathogen transmission pathways to control and / or prevent the spread of infection.

A Complex of Badnavirus Species Infecting Cacao Reveals Mixed Infections, Extensive Genomic Variability, and Interspecific Recombination.

The incidence of cacao swollen shoot disease (CSSD) in cacao (Theobroma cacao L.) has increased in West Africa since ~2000. To investigate the genomic and species diversity of the CSSD-badnaviruses infecting cacao in Côte d’Ivoire and Ghana, symptomatic leaves were subjected to high-throughput sequencing. Among the 30 newly determined genomes, three badnaviruses were identified, Cacao swollen shoot Togo B virus (CSSTBV), Cacao swollen shoot CD virus, and Cacao swollen shoot CE virus (CSSCEV). The phylogenetic trees reconstructed for the reverse transcriptase (RT) and ribonuclease H (RNase H) sequences were incongruent with the complete viral genomes, which had the most robust statistical support. Recombination seems to be involved in the CSSD-badnavirus diversification. The genomic diversity varied among different CSSD-badnaviruses, with CSSTBV showing the lowest nucleotide diversity (π = 0.06236), and CSSCEV exhibiting the greatest variability (π = 0.21911). Evidence of strong purifying selection was found in the coding regions of the CSSTBV isolates.

Sequencing and phylogenetic characterization of a novel RNA virus genome from Harmonia axyridis.

As a natural predator of many insect pests on its native Asian range, Harmonia axyridis remains amongst the insects whose pathogenic or beneficial microorganisms are yet to be studied. The genome nucleotide (nt) and amino acid sequences of open reading frames (ORFs) of the novel RNA virus were identified. Neighbor-joining (NJ) were constructed using MEGA7 software packages with nt sequences and conserved amino acid sequences of predicted RNA-dependent RNA polymerase (RdRp).The complete genome of a novel virus named Harmonia axyridis virus 1 was determined by RNA-seq and rapid amplification of cDNA ends from H. axyridis, which had a single-stranded RNA genome of 8868 nts in length and contains two putative ORFs. ORF1 encodes a polypeptide of 2182 amino acids, which contained conserved domains for 2 picornavirus-like capsid proteins and one RNA helicase. ORF2 encodes a polypeptide of 655 amino acids, which contained 1 RdRp domain. Phylogenetic analysis of whole genome nt sequences and RdRp deduced amino acid sequences suggested that the virus clustered with several unclassified Hubei picorna-like virus. To our knowledge, this is the first full annotated genome of a novel member of the unclassified group of RNA viruses, infecting H. axyridis in natural field conditions.

Detection of RNA viruses in Cape gooseberry (Physalis peruviana L.) by RNAseq using total RNA and dsRNA inputs

Physalis peruviana L. is a herbaceous solanaceous plant native to the South American Andes that produces a small yellow fruit of about 1-2 cm in diameter with high demand in the international markets. Currently, Colombia is the largest worldwide exporter of this fruit crop but, due to the emergence of several phytosanitary problems, yields have experienced a steady declined in recent years. Viruses are not included in current disease management programs of P. peruviana but, evidence suggest that viruses might play an important role in decreasing the productivity of this crop. High-throughput sequencing (HTS) has allowed a deeper understanding of the P. peruviana genome and promises to become a routine technique in future integrated disease management programs. Here, we evaluate the influence of the RNA input (total RNA vs dsRNA) on the sensitivity of HTS as a virus detection tool. Our results suggest that, total RNA can produce a high number of viral sequences but fails to detect viruses present at low concentrations and detectable using dsRNA. In particular, infection by potato mop-top virus (PMTV) was detected using both inputs. In addition to PMTV, dsRNA also allowed the assembly of a complete Potato yellow vein virus (PYVV) genome and scaffolds of Potato yellowing virus (PYV), Southern tomato virus (STV) and Potato virus S (PVS). Evidence for the presence of Potato virus X (PVX), Potato virus V (PVV), Potato virus Y (PVY), Bell pepper alphaendornavirus (BPEV) and a putative mycovirus was also obtained from the dsRNA dataset.