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Article ; Online: Defective Interfering Particles of Negative-Strand RNA Viruses.

Ziegler, Christopher M / Botten, Jason W

2020 Volume 28, Issue 7, Page(s) 554–565

Abstract: Viral defective interfering particles (DIPs) were intensely studied several decades ago but research waned leaving open many critical questions. New technologies and other advances led to a resurgence in DIP studies for negative-strand RNA viruses. While ...

Abstract	Viral defective interfering particles (DIPs) were intensely studied several decades ago but research waned leaving open many critical questions. New technologies and other advances led to a resurgence in DIP studies for negative-strand RNA viruses. While DIPs have long been recognized, their exact contribution to the outcome of acute or persistent viral infections has remained elusive. Recent studies have identified defective viral genomes (DVGs) in human infections, including respiratory syncytial virus and influenza, and growing evidence indicates that DVGs influence disease severity and may contribute to viral persistence. Further, several studies have advanced our understanding of key viral and host factors that regulate DIP formation and activity. Here we review these discoveries and highlight key questions moving forward.
MeSH term(s)	Defective Viruses/genetics ; Gene Deletion ; Genome, Viral/genetics ; Orthomyxoviridae/genetics ; Respiratory Syncytial Viruses/genetics ; Viral Interference/genetics ; Virus Replication/genetics
Language	English
Publishing date	2020-03-26
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	1158963-2
ISSN	1878-4380 ; 0966-842X
ISSN (online)	1878-4380
ISSN	0966-842X
DOI	10.1016/j.tim.2020.02.006
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Article ; Online: Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment.

Klaus, Joseph P / Botten, Jason

Journal of visualized experiments : JoVE

2016 , Issue 109, Page(s) e53682

Abstract: Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled ...

Abstract	Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.
MeSH term(s)	Arenavirus/physiology ; Humans ; RNA, Viral/analysis ; Real-Time Polymerase Chain Reaction/methods ; Virus Attachment
Chemical Substances	RNA, Viral
Keywords	covid19
Language	English
Publishing date	2016-03-02
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Video-Audio Media
ISSN	1940-087X
ISSN (online)	1940-087X
DOI	10.3791/53682
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Article: Defective Interfering Particles of Negative-Strand RNA Viruses

Ziegler, Christopher M / Botten, Jason W

Trends in microbiology. 2020,

2020

Abstract	Viral defective interfering particles (DIPs) were intensely studied several decades ago but research waned leaving open many critical questions. New technologies and other advances led to a resurgence in DIP studies for negative-strand RNA viruses. While DIPs have long been recognized, their exact contribution to the outcome of acute or persistent viral infections has remained elusive. Recent studies have identified defective viral genomes (DVGs) in human infections, including respiratory syncytial virus and influenza, and growing evidence indicates that DVGs influence disease severity and may contribute to viral persistence. Further, several studies have advanced our understanding of key viral and host factors that regulate DIP formation and activity. Here we review these discoveries and highlight key questions moving forward.
Keywords	Respiratory syncytial virus ; disease severity ; genome ; human diseases ; influenza
Language	English
Publishing place	Elsevier Ltd
Document type	Article
Note	Pre-press version
ZDB-ID	1158963-2
ISSN	1878-4380 ; 0966-842X
ISSN (online)	1878-4380
ISSN	0966-842X
DOI	10.1016/j.tim.2020.02.006
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Article ; Online: Laboratory Worker Self-Contamination with Noninfectious SARS-CoV-2 DNA Can Result in False-Positive Reverse Transcriptase PCR-Based Surveillance Testing.

Montgomery, Theresa L / Paavola, Michelle / Bruce, Emily A / Botten, Jason W / Crothers, Jessica W / Krementsov, Dimitry N

Journal of clinical microbiology

2021 Volume 59, Issue 7, Page(s) e0072321

MeSH term(s)	COVID-19 ; Clinical Laboratory Techniques ; DNA ; Humans ; Laboratories ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2021-06-18
Publishing country	United States
Document type	Letter
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/JCM.00723-21
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Zs.A 1188: Show issues

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Article ; Online: SARS-CoV-2 triggers DNA damage response in Vero E6 cells.

Victor, Joshua / Deutsch, Jamie / Whitaker, Annalis / Lamkin, Erica N / March, Anthony / Zhou, Pei / Botten, Jason W / Chatterjee, Nimrat

Biochemical and biophysical research communications

2021 Volume 579, Page(s) 141–145

Abstract: The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and ... ...

Abstract	The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response.
MeSH term(s)	Animals ; Ataxia Telangiectasia Mutated Proteins/genetics ; COVID-19/genetics ; Checkpoint Kinase 1/metabolism ; Chlorocebus aethiops ; DNA Damage ; Histones/genetics ; Host-Pathogen Interactions/genetics ; Phosphorylation ; SARS-CoV-2/pathogenicity ; Telomere ; Vero Cells
Chemical Substances	Histones ; Ataxia Telangiectasia Mutated Proteins (EC 2.7.11.1) ; Checkpoint Kinase 1 (EC 2.7.11.1)
Language	English
Publishing date	2021-09-15
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	205723-2
ISSN	1090-2104 ; 0006-291X ; 0006-291X
ISSN (online)	1090-2104 ; 0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2021.09.024
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Zs.A 116: Show issues

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Article ; Online: Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs.

Haist, Kelsey / Ziegler, Christopher / Botten, Jason

PloS one

2015 Volume 10, Issue 5, Page(s) e0120043

Abstract: Arenaviruses are bi-segmented, single-stranded RNA viruses that cause significant human disease. The manner in which they regulate the replication of their genome is not well-understood. This is partly due to the absence of a highly sensitive assay to ... ...

Abstract	Arenaviruses are bi-segmented, single-stranded RNA viruses that cause significant human disease. The manner in which they regulate the replication of their genome is not well-understood. This is partly due to the absence of a highly sensitive assay to measure individual species of arenavirus replicative RNAs. To overcome this obstacle, we designed a quantitative reverse transcription (RT)-PCR assay for selective quantitation of each of the lymphocytic choriomeningitis virus (LCMV) genomic or antigenomic RNAs. During the course of assay design, we identified a nonspecific priming phenomenon whereby, in the absence of an RT primer, cDNAs complementary to each of the LCMV replicative RNA species are generated during RT. We successfully circumvented this nonspecific priming event through the use of biotinylated primers in the RT reaction, which permitted affinity purification of primer-specific cDNAs using streptavidin-coated magnetic beads. As proof of principle, we used the assay to map the dynamics of LCMV replication at acute and persistent time points and to determine the quantities of genomic and antigenomic RNAs that are incorporated into LCMV particles. This assay can be adapted to measure total S or L segment-derived viral RNAs and therefore represents a highly sensitive diagnostic platform to screen for LCMV infection in rodent and human tissue samples and can also be used to quantify virus-cell attachment.
MeSH term(s)	Arenaviridae/genetics ; DNA, Complementary/genetics ; Genome, Viral/genetics ; RNA, Viral/genetics ; Reverse Transcriptase Polymerase Chain Reaction/methods
Chemical Substances	DNA, Complementary ; RNA, Viral
Keywords	covid19
Language	English
Publishing date	2015-05-15
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0120043
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Highly sensitive assay for measurement of arenavirus-cell attachment

Klaus, Joseph P / Botten, Jason

Journal of visualized experiments. 2016 Mar. 02, , no. 109

2016

Abstract	Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.
Keywords	quantitative polymerase chain reaction ; human diseases ; radionuclides ; virion ; genome ; reverse transcriptase polymerase chain reaction ; Argentinian mammarenavirus ; endocytosis ; viruses ; genomics ; biotin ; RNA ; fluorescent dyes ; covid19
Language	English
Dates of publication	2016-0302
Size	p. e53682.
Publishing place	Journal of Visualized Experiments
Document type	Article
Note	2019-12-06
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/53682
Database	NAL-Catalogue (AGRICOLA)

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Article: SARS-CoV-2 triggers DNA damage response in Vero E6 cells

Victor, Joshua / Deutsch, Jamie / Whitaker, Annalis / Lamkin, Erica N. / March, Anthony / Zhou, Pei / Botten, Jason W. / Chatterjee, Nimrat

Biochemical and biophysical research communications. 2021 Nov. 19, v. 579

2021

Abstract	The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response.
Keywords	COVID-19 infection ; Cercopithecus aethiops ; DNA damage ; Severe acute respiratory syndrome coronavirus 2 ; immune response ; kidneys ; phosphorylation ; research ; telomeres ; transcription (genetics) ; viruses
Language	English
Dates of publication	2021-1119
Size	p. 141-145.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2021.09.024
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Article ; Online: Clinical Performance Characteristics of the Swift Normalase Amplicon Panel for Sensitive Recovery of Severe Acute Respiratory Syndrome Coronavirus 2 Genomes.

Shrestha, Lasata / Lin, Michelle J / Xie, Hong / Mills, Margaret G / Mohamed Bakhash, Shah A / Gaur, Vinod P / Livingston, Robert J / Castor, Jared / Bruce, Emily A / Botten, Jason W / Huang, Meei-Li / Jerome, Keith R / Greninger, Alexander L / Roychoudhury, Pavitra

The Journal of molecular diagnostics : JMD

2022 Volume 24, Issue 9, Page(s) 963–976

Abstract: Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the ... ...

Abstract	Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of C
MeSH term(s)	COVID-19/genetics ; Genome, Viral ; Humans ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; Whole Genome Sequencing/methods
Chemical Substances	RNA, Viral
Language	English
Publishing date	2022-07-18
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2000060-1
ISSN	1943-7811 ; 1525-1578
ISSN (online)	1943-7811
ISSN	1525-1578
DOI	10.1016/j.jmoldx.2022.05.007
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Zs.A 5146: Show issues

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Article ; Online: Pathogenic human variant that dislocates GATA2 zinc fingers disrupts hematopoietic gene expression and signaling networks.

The Journal of clinical investigation

2023 Volume 133, Issue 7

Abstract: Although certain human genetic variants are conspicuously loss of function, decoding the impact of many variants is challenging. Previously, we described a patient with leukemia predisposition syndrome (GATA2 deficiency) with a germline GATA2 variant ... ...

Abstract	Although certain human genetic variants are conspicuously loss of function, decoding the impact of many variants is challenging. Previously, we described a patient with leukemia predisposition syndrome (GATA2 deficiency) with a germline GATA2 variant that inserts 9 amino acids between the 2 zinc fingers (9aa-Ins). Here, we conducted mechanistic analyses using genomic technologies and a genetic rescue system with Gata2 enhancer-mutant hematopoietic progenitor cells to compare how GATA2 and 9aa-Ins function genome-wide. Despite nuclear localization, 9aa-Ins was severely defective in occupying and remodeling chromatin and regulating transcription. Variation of the inter-zinc finger spacer length revealed that insertions were more deleterious to activation than repression. GATA2 deficiency generated a lineage-diverting gene expression program and a hematopoiesis-disrupting signaling network in progenitors with reduced granulocyte-macrophage colony-stimulating factor (GM-CSF) and elevated IL-6 signaling. As insufficient GM-CSF signaling caused pulmonary alveolar proteinosis and excessive IL-6 signaling promoted bone marrow failure and GATA2 deficiency patient phenotypes, these results provide insight into mechanisms underlying GATA2-linked pathologies.
MeSH term(s)	Humans ; Granulocyte-Macrophage Colony-Stimulating Factor ; GATA2 Deficiency/genetics ; Interleukin-6/genetics ; Hematopoiesis/genetics ; Gene Expression ; Zinc Fingers/genetics ; GATA2 Transcription Factor/genetics ; GATA2 Transcription Factor/metabolism
Chemical Substances	Granulocyte-Macrophage Colony-Stimulating Factor (83869-56-1) ; Interleukin-6 ; GATA2 Transcription Factor ; GATA2 protein, human
Language	English
Publishing date	2023-04-03
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	3067-3
ISSN	1558-8238 ; 0021-9738
ISSN (online)	1558-8238
ISSN	0021-9738
DOI	10.1172/JCI162685
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