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Sunday, July 7, 2019

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into…

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Sunday, July 7, 2019

Single-virion sequencing of lamivudine-treated HBV populations reveal population evolution dynamics and demographic history.

Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we…

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Sunday, July 7, 2019

Pathogenicity and whole genome sequence analysis of a Pseudorabies virus strain FJ-2012 isolated from Fujian, Southern China.

The outbreaks of pseudorabies have been frequently reported in Bartha-K61-vaccinated farms in China since 2011. To study the pathogenicity and evolution of the circulating pseudorabies viruses in Fujian Province, mainland China, we isolated and sequenced the whole genome of a wild-type pseudorabies virus strain named “FJ-2012.” We then conducted a few downstream bioinformatics analyses including phylogenetic analysis and pathogenic analysis and used the virus to infect 6 pseudorabies virus-free piglets. FJ-2012-infected piglets developed symptoms like high body temperature and central nervous system disorders and had high mortality rate. In addition, we identified typical micropathological changes such as multiple gross lesions…

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Sunday, July 7, 2019

Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus.

Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these…

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Sunday, July 7, 2019

Estimating fitness of viral quasispecies from next-generation sequencing data.

The quasispecies model is ubiquitous in the study of viruses. While having lead to a number of insights that have stood the test of time, the quasispecies model has mostly been discussed in a theoretical fashion with little support of data. With next-generation sequencing (NGS), this situation is changing and a wealth of data can now be produced in a time- and cost-efficient manner. NGS can, after removal of technical errors, yield an exceedingly detailed picture of the viral population structure. The widespread availability of cross-sectional data can be used to study fitness landscapes of viral populations in the quasispecies…

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Sunday, July 7, 2019

Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing.

Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naïve-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naïve patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule…

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Sunday, July 7, 2019

Genome of Cnaphalocrocis medinalis granulovirus, the first Crambidae-infecting betabaculovirus isolated from rice leaffolder to sequenced.

Cnaphalocrocis medinalis is a major pest of rice in South and South-East Asia. Insecticides are the major means farmers use for management. A naturally occurring baculovirus, C. medinalis granulovirus (CnmeGV), has been isolated from the larvae and this has the potential for use as microbial agent. Here, we described the complete genome sequence of CnmeGV and compared it to other baculovirus genomes. The genome of CnmeGV is 112,060 base pairs in length, has a G+C content of 35.2%. It contains 133 putative open reading frames (ORFs) of at least 150 nucleotides. A hundred and one (101) of these ORFs are…

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Sunday, July 7, 2019

Genomic analyses reveal that partial sequence of an earlier pseudorabies virus in China is originated from a Bartha-vaccine-like strain.

Pseudorabies virus (PRV), the causative agent of Aujeszky?s disease, has gained increased attention in China in recent years as a result of the outbreak of emergent pseudorabies. Several genomic and partial sequences are available for Chinese emergent and European-American strains of PRV, but limited sequence data exist for the earlier Chinese strains. In this study, we determined the complete genomic sequence of one earlier Chinese strain SC and one emergent strain HLJ8. Compared with other known sequences, we demonstrated that PRV strains from distinct geographical regions displayed divergent evolution. Additionally, we report for the first time, a recombination event between…

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Sunday, July 7, 2019

A strange endocytobiont revealed as largest virus.

A lot of endocytobionts (or endosymbionts) have been discovered within free-living amoebae in recent years. In this article the results of a long lasting effort to derive valuable data about an extraordinary spore-like infectious microorganism (endocytobiont, endosymbiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens case of a patient with keratitis, are presented. It took some time until this endocytobiont could be attributed to the genus Pandoravirus following a publication of two other pandoraviruses isolated from aquatic environments. Consequently the molecular biological investigation led to the taxonomic affiliation of the endocytobiont with the genus Pandoravirus and to…

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Sunday, July 7, 2019

Evolution of coreceptor utilization to escape CCR5 antagonist therapy.

The HIV-1 envelope interacts with coreceptors CCR5 and CXCR4 in a dynamic, multi-step process, its molecular details not clearly delineated. Use of CCR5 antagonists results in tropism shift and therapeutic failure. Here we describe a novel approach using full-length patient-derived gp160 quasispecies libraries cloned into HIV-1 molecular clones, their separation based on phenotypic tropism in vitro, and deep sequencing of the resultant variants for structure-function analyses. Analysis of functionally validated envelope sequences from patients who failed CCR5 antagonist therapy revealed determinants strongly associated with coreceptor specificity, especially at the gp120-gp41 and gp41-gp41 interaction surfaces that invite future research on the…

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Sunday, July 7, 2019

Complete genome sequence of pseudorabies virus reference strain NIA3 using single-molecule real-time sequencing.

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease in pigs. PRV strains are also used as model organisms for the study of alphaherpesvirus biology or for neuronal pathway studies. We present here the complete genome of the virulent wild-type PRV reference strain NIA3, determined by single-molecule real-time sequencing. Copyright © 2016 Mathijs et al.

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Sunday, July 7, 2019

Long single-molecule reads can resolve the complexity of the influenza virus composed of rare, closely related mutant variants

As a result of a high rate of mutations and recombination events, an RNA-virus exists as a heterogeneous “swarm” of mutant variants. The long read length offered by single-molecule sequencing technologies allows each mutant variant to be sequenced in a single pass. However, high error rate limits the ability to reconstruct heterogeneous viral population composed of rare, related mutant variants. In this paper, we present 2SNV, a method able to tolerate the high error-rate of the single-molecule protocol and reconstruct mutant variants. 2SNV uses linkage between single nucleotide variations to efficiently distinguish them from read errors. To benchmark the sensitivity…

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Sunday, July 7, 2019

Chloroplast genomes: diversity, evolution, and applications in genetic engineering.

Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. We also discuss the potential biotechnological applications of chloroplast genomes.

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Sunday, July 7, 2019

Next-generation sequencing: a diagnostic one-stop shop for Hepatitis C?

Before starting chronic hepatitis C treatment, the viral genotype/subtype has to be accurately determined and potentially coupled with drug resistance testing. Due to the high genetic variability of the hepatitis C virus, this can be a demanding task that can potentially be streamlined by viral whole-genome sequencing using next-generation sequencing as demonstrated by an article in this issue of the Journal of Clinical Microbiology by E. Thomson, C. L. C. Ip, A. Badhan, M. T. Christiansen, W. Adamson, et al. (J Clin Microbiol. 54:2455-2469, 2016, http://dx.doi.org/10.1128/JCM.00330-16). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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