PMID- 33191939
OWN - NLM
STAT- MEDLINE
DCOM- 20201225
LR  - 20211030
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 164
DP  - 2020 Oct 29
TI  - Single-cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and 
      Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection.
LID - 10.3791/61843 [doi]
AB  - Capturing the dynamic replication and assembly processes of viruses has been 
      hindered by the lack of robust in situ hybridization (ISH) technologies that 
      enable sensitive and simultaneous labeling of viral nucleic acid and protein. 
      Conventional DNA fluorescence in situ hybridization (FISH) methods are often not 
      compatible with immunostaining. We have therefore developed an imaging approach, 
      MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and 
      protein), which enables simultaneous single-cell visualization of DNA, RNA, and 
      protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) 
      ISH technology, which dramatically improves oligonucleotide probe sensitivity and 
      detection. Small modifications of MICDDRP enable imaging of viral proteins 
      concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have 
      applied these protocols to study the life cycles of multiple viral pathogens, 
      including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus 
      (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and 
      influenza A virus (IAV). We demonstrated that we can efficiently label viral 
      nucleic acids and proteins across a diverse range of viruses. These studies can 
      provide us with improved mechanistic understanding of multiple viral systems, and 
      in addition, serve as a template for application of multiplexed fluorescence 
      imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.
FAU - Shah, Raven
AU  - Shah R
AD  - Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory 
      University School of Medicine.
FAU - Lan, Shuiyun
AU  - Lan S
AD  - Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory 
      University School of Medicine.
FAU - Puray-Chavez, Maritza N
AU  - Puray-Chavez MN
AD  - Department of Molecular Microbiology & Immunology, University of Missouri School 
      of Medicine.
FAU - Liu, Dandan
AU  - Liu D
AD  - Department of Molecular Microbiology & Immunology, University of Missouri School 
      of Medicine.
FAU - Tedbury, Philip R
AU  - Tedbury PR
AD  - Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory 
      University School of Medicine.
FAU - Sarafianos, Stefan G
AU  - Sarafianos SG
AD  - Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory 
      University School of Medicine; stefanos.sarafianos@emory.edu.
LA  - eng
GR  - R01 AI120860/AI/NIAID NIH HHS/United States
GR  - R01 AI148382/AI/NIAID NIH HHS/United States
GR  - R37 AI076119/AI/NIAID NIH HHS/United States
GR  - R01 AI121315/AI/NIAID NIH HHS/United States
GR  - R01 AI146017/AI/NIAID NIH HHS/United States
GR  - U54 AI150472/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Video-Audio Media
DEP - 20201029
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (DNA, Viral)
RN  - 0 (RNA, Viral)
RN  - 0 (Viral Proteins)
SB  - IM
MH  - DNA, Viral/*analysis/genetics
MH  - HIV-1/genetics
MH  - Hepacivirus/genetics
MH  - Hepatitis B virus/genetics
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - *Optical Imaging
MH  - Orthomyxoviridae/genetics
MH  - RNA, Viral/*analysis/genetics
MH  - *Single-Cell Analysis
MH  - Viral Proteins/*analysis
MH  - Virus Diseases/*diagnosis/*genetics
MH  - Zika Virus/genetics
PMC - PMC7774762
MID - NIHMS1655199
EDAT- 2020/11/17 06:00
MHDA- 2020/12/29 06:00
PMCR- 2021/10/29
CRDT- 2020/11/16 08:42
PHST- 2020/11/16 08:42 [entrez]
PHST- 2020/11/17 06:00 [pubmed]
PHST- 2020/12/29 06:00 [medline]
PHST- 2021/10/29 00:00 [pmc-release]
AID - 10.3791/61843 [doi]
PST - epublish
SO  - J Vis Exp. 2020 Oct 29;(164):10.3791/61843. doi: 10.3791/61843.