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      A Fosmid-Based System for the Generation of Recombinant Cercopithecine Alphaherpesvirus 2 Encoding Reporter Genes

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          Abstract

          The transmission of Macacine alphaherpesvirus 1 (McHV-1) from macaques, the natural host, to humans causes encephalitis. In contrast, human infection with Cercopithecine alphaherpesvirus 2 (CeHV-2), a closely related alphaherpesvirus from African vervet monkeys and baboons, has not been reported and it is believed that CeHV-2 is apathogenic in humans. The reasons for the differential neurovirulence of McHV-1 and CeHV-2 have not been explored on a molecular level, in part due to the absence of systems for the production of recombinant viruses. Here, we report the generation of a fosmid-based system for rescue of recombinant CeHV-2. Moreover, we show that, in this system, recombineering can be used to equip CeHV-2 with reporter genes. The recombinant CeHV-2 viruses replicated with the same efficiency as uncloned, wt virus and allowed the identification of cell lines that are highly susceptible to CeHV-2 infection. Collectively, we report a system that allows rescue and genetic modification of CeHV-2 and likely other alphaherpesviruses. This system should aid future analysis of CeHV-2 biology.

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          Most cited references26

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          Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome.

          A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete construction of a mutant herpesvirus genome can now be carried out in a controlled manner prior to the reconstitution of infectious progeny. The described approach should be generally applicable to the mutagenesis of genomes of other large DNA viruses.
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            Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.

            The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome
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              Evolutionary Origins of Human Herpes Simplex Viruses 1 and 2

              Herpesviruses have been infecting and codiverging with their vertebrate hosts for hundreds of millions of years. The primate simplex viruses exemplify this pattern of virus–host codivergence, at a minimum, as far back as the most recent common ancestor of New World monkeys, Old World monkeys, and apes. Humans are the only primate species known to be infected with two distinct herpes simplex viruses: HSV-1 and HSV-2. Human herpes simplex viruses are ubiquitous, with over two-thirds of the human population infected by at least one virus. Here, we investigated whether the additional human simplex virus is the result of ancient viral lineage duplication or cross-species transmission. We found that standard phylogenetic models of nucleotide substitution are inadequate for distinguishing among these competing hypotheses; the extent of synonymous substitutions causes a substantial underestimation of the lengths of some of the branches in the phylogeny, consistent with observations in other viruses (e.g., avian influenza, Ebola, and coronaviruses). To more accurately estimate ancient viral divergence times, we applied a branch-site random effects likelihood model of molecular evolution that allows the strength of natural selection to vary across both the viral phylogeny and the gene alignment. This selection-informed model favored a scenario in which HSV-1 is the result of ancient codivergence and HSV-2 arose from a cross-species transmission event from the ancestor of modern chimpanzees to an extinct Homo precursor of modern humans, around 1.6 Ma. These results provide a new framework for understanding human herpes simplex virus evolution and demonstrate the importance of using selection-informed models of sequence evolution when investigating viral origin hypotheses.
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                Author and article information

                Journal
                Viruses
                Viruses
                viruses
                Viruses
                MDPI
                1999-4915
                05 November 2019
                November 2019
                : 11
                : 11
                : 1026
                Affiliations
                [1 ]Infection Biology Unit, German Primate Center-Leibniz Institute for Primate Research, 37077 Göttingen, Germany; ekaterina.chukhno@ 123456gmail.com (E.C.); SGaertner@ 123456dpz.eu (S.G.); ASiregar@ 123456dpz.eu (A.R.S.); alex.mehr@ 123456freenet.de (A.M.); m.wende@ 123456tu-braunschweig.de (M.W.); spoehlmann@ 123456dpz.eu (S.P.)
                [2 ]Faculty of Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
                [3 ]Platform Degenerative Diseases, German Primate Center-Leibniz Institute for Primate Research, 37077 Göttingen, Germany; SPetkov@ 123456dpz.eu
                [4 ]German Center for Cardiovascular Research (DZHK), partner site, 37099 Göttingen, Germany
                [5 ]Institute of Virology, Hannover Medical School, 30625 Hannover, Germany; Goetting.Jasper@ 123456mh-hannover.de (J.G.); Dhingra.Akshay@ 123456mh-hannover.de (A.D.); Schulz.Thomas@ 123456mh-hannover.de (T.S.)
                [6 ]Faculty of Biology and Psychology, University Göttingen, 37073 Göttingen, Germany
                Author notes
                [* ]Correspondence: mwinkler@ 123456dpz.eu ; Tel.: +49-551-3851383
                Author information
                https://orcid.org/0000-0002-3891-5746
                https://orcid.org/0000-0001-6086-9136
                Article
                viruses-11-01026
                10.3390/v11111026
                6893520
                31694178
                9f266308-895c-4941-bed6-c9cbc9fe8ee7
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 23 September 2019
                : 31 October 2019
                Categories
                Article

                Microbiology & Virology
                cercopithecine alphaherpesvirus 2,fosmid,recombineering
                Microbiology & Virology
                cercopithecine alphaherpesvirus 2, fosmid, recombineering

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