In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome

Autor: Travis Whitmer, Jason Shao, Scott G. Hansen, Richard J. Stanton, Xiaofei E, Paul T. Edlefsen, Lesli M. Sprehe, Hillary C. Cleveland-Rubeor, Amitinder Kaur, Matilda J. Moström, Matthew R. McArdle, Colette M. Hughes, Peter A. Barry, Daniel Malouli, Andrea N. Selseth, Michael Nekorchuk, Eisa Mahyari, Jacob D. Estes, Michael K. Axthelm, Kerianne A. Jackson, Craig N. Kreklywich, Daniel N. Streblow, Yujuan Yue, Timothy F. Kowalik, Jeremy Smedley, Klaus Früh, Husam Taher, Benjamin N. Bimber, Luke S. Uebelhoer, Amruta Bhusari, Dawn L. Roberts, Louis J. Picker, Kimberli A. Schmidt, Elizabeth A. Scheef, Abigail B. Ventura
Přispěvatelé: Kalejta, Robert F
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Cytomegalovirus Infection
Male
Viral Diseases
Chromosomes
Artificial
Bacterial
Physiology
Cytomegalovirus
Urine
Monkeys
Genome
Biochemistry
Recombineering
Medical Conditions
Animal Cells
Medicine and Health Sciences
2.2 Factors relating to the physical environment
Viral
Biology (General)
Aetiology
Phylogeny
Connective Tissue Cells
Mammals
Recombinant
Mammalian Genomics
Bacterial
Eukaryota
Genomics
Body Fluids
Nucleic acids
Infectious Diseases
Medical Microbiology
Connective Tissue
Artificial
Vertebrates
Cytomegalovirus Infections
Female
Cellular Types
Anatomy
Infection
Macaque
Biotechnology
Research Article
Primates
QH301-705.5
Gene prediction
Immunology
DNA
Recombinant
DNA repair
Viremia
Genome
Viral
Biology
Microbiology
Virus
Chromosomes
Cell Line
Open Reading Frames
Species Specificity
In vivo
Virology
Old World monkeys
medicine
Genetics
Animals
Humans
Gene Prediction
Molecular Biology
Gene
Bacterial artificial chromosome
Animal
Organisms
Biology and Life Sciences
Computational Biology
Cell Biology
DNA
RC581-607
Fibroblasts
medicine.disease
Genome Analysis
Macaca mulatta
Disease Models
Animal
Biological Tissue
Animal Genomics
Disease Models
Amniotes
Mutation
Parasitology
Immunologic diseases. Allergy
Zoology
Zdroj: PLoS Pathogens
PLoS pathogens, vol 16, iss 11
PLoS Pathogens, Vol 16, Iss 11, p e1008666 (2020)
ISSN: 1553-7374
1553-7366
Popis: Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68–1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68–1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68–1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68–1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68–1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.
Author summary Human cytomegalovirus (HCMV) infections are generally asymptomatic in healthy immunocompetent individuals, but HCMV can cause serious disease after congenital infection and in individuals with immunocompromised immune systems. Since HCMV is highly species specific and cannot productively infect immunocompetent laboratory animals, experimental infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines against chronic and recurring infections with pathogens such as the human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted with derivatives of the 68–1 strain of RhCMV which has acquired multiple genomic alterations in tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naïve RM with FL-RhCMV demonstrated viremia and tissue dissemination that was comparable to that of non-clonal low passage isolates. We further demonstrate that FL-RhCMV is strongly attenuated upon deletion of gene regions absent in 68–1 thus demonstrating the usefulness of FL-RhCMV to study RhCMV pathogenesis.
Databáze: OpenAIRE
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