Genomic and in silico Structural Characterization of Dobrava-Belgrade orthohantavirus Isolate from European Side of Turkey

Abstract Orthohantaviruses are transmitted to humans mostly through small mammals that are the reservoirs of these viruses. Because orthohantaviruses show high genetic variability through geographic regions, the genetic characterization of these viruses with whole genome sequencing is of great importance to clarify the molecular epidemiology and track their genetic changes in the reservoir hosts. We have previously reported the presence of Dobrava-Belgrade orthohantavirus (DOBV) in the Igneada region, Kirklareli province by showing antibodies against the virus in rodents and by sequencing partial genomes of the virus. Here we report the whole genome sequencing of DOBV Igneada strain directly from Apodemus flavicollis’ lung tissue by next-generation sequencing followed by phylogenetic analyses. In addition, viral protein structures of DOBV Igneada strain were modelled, and in silico prediction analyses of amino acid changes on viral protein function and stability were performed.The phylogenetic analysis showed a close relation between the DOBV Igneada strain from Turkey and DOBV Ano-Poroia strain from Greece. Similarity plot analysis revealed also similarities between DOBV Igneada strain and other DOBV strains from the Balkans such as Greece, Croatia, and Slovenia. Additionally, in silico prediction suggested that G318E, Y322H, and S324P mutations on Gn glycoprotein are deleterious, and all amino acid changes decrease the stability of both Gn and Gc glycoproteins.In conclusion, full orthohantaviral genomes can be obtained directly from rodent lung tissues allowing detailed genetic and structural analyses of orthohantaviruses. The DOBV Igneada strain shows great similarity to the prototype Ano-Poroia strain, yet it was predicted that DOBV Igneada strain may have some changes on its pathogenicity and its structure warranting further research.

Download Full-text

In Silico Genotyping of Escherichia coli Isolates for Extraintestinal Virulence Genes by Use of Whole-Genome Sequencing Data

Journal of Clinical Microbiology ◽

10.1128/jcm.01269-20 ◽

2020 ◽

Vol 58 (10) ◽

Author(s):

Anna Maria Malberg Tetzschner ◽

James R. Johnson ◽

Brian D. Johnston ◽

Ole Lund ◽

Flemming Scheutz

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

In Silico ◽

Virulence Genes ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Content Type ◽

E Coli

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause in humans of urinary tract infection and bacteremia. The previously published web tool VirulenceFinder (http://cge.cbs.dtu.dk/services/VirulenceFinder/) uses whole-genome sequencing (WGS) data for in silico characterization of E. coli isolates and enables researchers and clinical health personnel to quickly extract and interpret virulence-relevant information from WGS data. In this study, 38 ExPEC-associated virulence genes were added to the existing E. coli VirulenceFinder database. In total, 14,441 alleles were downloaded. A total of 1,890 distinct alleles were added to the database after removal of redundant sequences and analysis of the remaining alleles for open reading frames (ORFs). The database now contains 139 genes—of which 44 are related to ExPEC—and 2,826 corresponding alleles. Construction of the database included validation against 27 primer pairs from previous studies, a search for serotype-specific P fimbriae papA alleles, and a BLASTn confirmation of seven genes (etsC, iucC, kpsE, neuC, sitA, tcpC, and terC) not covered by the primers. The augmented database was evaluated using (i) a panel of nine control strains and (ii) 288 human-source E. coli strains classified by PCR as ExPEC and non-ExPEC. We observed very high concordance (average, 93.4%) between PCR and WGS findings, but WGS identified more alleles. In conclusion, the addition of 38 ExPEC-associated genes and the associated alleles to the E. coli VirulenceFinder database allows for a more complete characterization of E. coli isolates based on WGS data, which has become increasingly important considering the plasticity of the E. coli genome.

Download Full-text

Faculty Opinions recommendation of Characterization of uterine leiomyomas by whole-genome sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718021966.793479049 ◽

2013 ◽

Author(s):

Hiroshi Ishikawa

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Uterine Leiomyomas ◽

Whole Genome

Download Full-text

Characterization of Shiga Toxin‐Producing Escherichia coli Isolated From Cattle and Sheep in Xinjiang Province, China, Using Whole‐Genome Sequencing

Transboundary and Emerging Diseases ◽

10.1111/tbed.13999 ◽

2021 ◽

Author(s):

Yingyu Liu ◽

Huoming Li ◽

Xuhua Chen ◽

Panpan Tong ◽

Yan Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Shiga Toxin ◽

Whole Genome ◽

Cattle And Sheep

Download Full-text

SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

Scientific Reports ◽

10.1038/s41598-019-55144-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Weili Cai ◽

Schyler Nunziata ◽

John Rascoe ◽

Michael J. Stulberg

Keyword(s):

Whole Genome Sequencing ◽

Molecular Characterization ◽

Genome Sequencing ◽

Whole Genome Sequence ◽

Whole Genome ◽

Genome Coverage ◽

Metagenomic Sample ◽

Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

Download Full-text

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

10.21007/etd.cghs.2021.0551 ◽

2021 ◽

Author(s):

◽

Mariah Taylor ◽

Keyword(s):

Genetic Variation ◽

Amino Acid ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Rna Virus ◽

Animal Health ◽

Functional Domains ◽

Whole Genome ◽

Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

Download Full-text

Genetic Characterization of Brucella spp.: Whole Genome Sequencing-Based Approach for the Determination of Multiple Locus Variable Number Tandem Repeat Profiles

Frontiers in Microbiology ◽

10.3389/fmicb.2021.740068 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ana Pelerito ◽

Alexandra Nunes ◽

Teresa Grilo ◽

Joana Isidro ◽

Catarina Silva ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

In Silico ◽

Variable Number Tandem Repeat ◽

Epidemiological Studies ◽

Variable Number ◽

Epidemiological Surveillance ◽

Future Application ◽

Whole Genome

Brucellosis is an important zoonosis that is emerging in some regions of the world, gaining increased relevance with the inclusion of the causing agent Brucella spp. in the class B bioterrorism group. Until now, multi-locus VNTR Analysis (MLVA) based on 16 loci has been considered as the gold standard for Brucella typing. However, this methodology is laborious, and, with the rampant release of Brucella genomes, the transition from the traditional MLVA to whole genome sequencing (WGS)-based typing is on course. Nevertheless, in order to avoid a disruptive transition with the loss of massive genetic data obtained throughout the last decade and considering that the transition timings will vary considerably among different countries, it is important to determine WGS-based MLVA alleles of the nowadays sequenced genomes. On this regard, we aimed to evaluate the performance of a Python script that had been previously developed for the rapid in silico extraction of the MLVA alleles, by comparing it to the PCR-based MLVA procedure over 83 strains from different Brucella species. The WGS-based MLVA approach detected 95.3% of all possible 1,328 hits (83 strains×16 loci) and showed an agreement rate with the PCR-based MLVA procedure of 96.4% for MLVA-16. According to our dataset, we suggest the use of a minimal depth of coverage of ~50x and a maximum number of ~200 contigs as guiding “boundaries” for the future application of the script. In conclusion, the evaluated script seems to be a very useful and robust tool for the in silico determination of MLVA profiles of Brucella strains, allowing retrospective and prospective molecular epidemiological studies, which are important for maintaining an active epidemiological surveillance of brucellosis.

Download Full-text

Peer Review #1 of "Challenging a bioinformatic tool’s ability to detect microbial contaminants using in silico whole genome sequencing data (v0.2)"

10.7287/peerj.3729v0.2/reviews/1 ◽

2017 ◽

Author(s):

S Kumar

Keyword(s):

Whole Genome Sequencing ◽

Peer Review ◽

Genome Sequencing ◽

In Silico ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Microbial Contaminants

Download Full-text

First isolation and characterization of Brucella suis from yak

Genome ◽

10.1139/gen-2019-0101 ◽

2020 ◽

Vol 63 (8) ◽

pp. 397-405

Author(s):

Xiaowen Yang ◽

Ning Wang ◽

Xiaofang Cao ◽

Pengfei Bie ◽

Zhifeng Xing ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Health And Safety ◽

Host Cells ◽

Whole Genome ◽

Genetic Characteristics ◽

Qinghai Province ◽

Isolation And Characterization ◽

Smooth Strain

Brucella spp., facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonosis, affect public health and safety. A Brucella strain was isolated from yak in Qinghai Province. To detect whether this isolate could cause an outbreak of brucellosis and to reveal its genetic characteristics, several typing and whole-genome sequencing methods were applied to identify its species and genetic characteristics. Phylogenetic analysis based on MLVA and whole-genome sequencing revealed the genetic characteristics of the isolated strain. The results showed that the isolated strain is a B. suis biovar 1 smooth strain, and this isolate was named B. suis QH05. The results of comparative genomics and MLVA showed that B. suis QH05 is not a vaccine strain. Comparison with other B. suis strains isolated from humans and animals indicated that B. suis QH05 may be linked to specific animal and human sources. In conclusion, B. suis QH05 does not belong to the Brucella epidemic species in China, and as the first isolation of B. suis from yak, this strain expands the host range of B. suis.

Download Full-text