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Peer-reviewedCC BY 4.0mBio

TriLock-FISH enables in situ discrimination of influenza cRNA and mRNA for single-cell analysis of viral replication and transcription

  • Lingkai Zhang1,2,3
  • Wei Chen1,2,3
  • Xinyu Gao1,2,3
  • Huaiyuan Cai1,2,3
  • Xu Zhou1,2,3
  • Rizwan Ullah1,2,3
  • Xinyi Pei1,2,3
  • Jinxia Dai1
  • Gang Cao1,2,3,4
  • Guiqing Peng1,2,3,4
  • Hongbo Zhou1,2,3,4
  • Wen Su1,2,3,5
5 affiliations
  1. 1National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University
  2. 2Hubei Hongshan Laboratory
  3. 3Frontiers Science Center for Animal Breeding and Sustainable Production
  4. 4The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University
  5. 5Shennongjia Science and Technology Innovation Center, Shennongjia Multidisciplinary Observation and Research Station of the Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University
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Abstract

Influenza A virus produces three RNA species—viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA)—whose synthesis and trafficking are spatially compartmentalized. However, the high sequence similarity between cRNA and mRNA hinders their distinction in situ , limiting understanding of the spatial organization of the viral replication-transcription dynamics. Here, triple-lock fluorescence in situ hybridization (TriLock-FISH) was developed to distinguish vRNA, cRNA, and mRNA in situ with high specificity. TriLock-FISH enabled a genome-wide, time-resolved single-cell atlas of RNA abundance and localization, revealing marked cell-to-cell variation in RNA abundance but relatively conserved spatial organization. cRNA was found to be predominantly nuclear, with a small but reproducible cytoplasmic fraction. Application to infected mouse lung tissue demonstrated simultaneous spatial detection of all viral RNA species. This approach provides a highly specific framework for single-cell analysis of RNA virus replication and transcription.

IMPORTANCE: Influenza virus relies on three closely related RNA molecules for replication and gene expression, yet distinguishing them inside infected cells has remained technically challenging. This limitation has hindered understanding of viral replication in space and time. We developed triple-lock fluorescence in situ hybridization, which enables simultaneous visualization of all three viral RNA species across all eight influenza genome segments in infected cells and tissues. Single-cell analysis revealed striking heterogeneity in viral RNA abundance across infected cells, whereas intracellular RNA localization patterns remained relatively conserved. Complementary RNA, a replication intermediate long thought to be strictly nuclear, was also detected at low levels in the cytoplasm. These findings provide new insight into influenza virus replication and establish a broadly applicable tool for studying RNA virus biology.

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Cite

BibTeXRIS
BibTeX
@article{zhang2026trilockfish,
  title = {{TriLock-FISH enables in situ discrimination of influenza cRNA and mRNA for single-cell analysis of viral replication and transcription}},
  author = {Lingkai Zhang and Wei Chen and Xinyu Gao and Huaiyuan Cai and Xu Zhou and Rizwan Ullah and Xinyi Pei and Jinxia Dai and Gang Cao and Guiqing Peng and Hongbo Zhou and Wen Su},
  journal = {mBio},
  year = {2026},
  month = oct,
  doi = {10.1128/mbio.01572-26},
  url = {https://journals.asm.org/doi/10.1128/mbio.01572-26},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - TriLock-FISH enables in situ discrimination of influenza cRNA and mRNA for single-cell analysis of viral replication and transcription
AU  - Lingkai Zhang
AU  - Wei Chen
AU  - Xinyu Gao
AU  - Huaiyuan Cai
AU  - Xu Zhou
AU  - Rizwan Ullah
AU  - Xinyi Pei
AU  - Jinxia Dai
AU  - Gang Cao
AU  - Guiqing Peng
AU  - Hongbo Zhou
AU  - Wen Su
PY  - 2026
DA  - 2026/10/22
JO  - mBio
DO  - 10.1128/mbio.01572-26
UR  - https://journals.asm.org/doi/10.1128/mbio.01572-26
AB  - Influenza A virus produces three RNA species—viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA)—whose synthesis and trafficking are spatially compartmentalized. However, the high sequence similarity between cRNA and mRNA hinders their distinction in situ , limiting understanding of the spatial organization of the viral replication-transcription dynamics. Here, triple-lock fluorescence in situ hybridization (TriLock-FISH) was developed to distinguish vRNA, cRNA, and mRNA in situ with high specificity. TriLock-FISH enabled a genome-wide, time-resolved single-cell atlas of RNA abundance and localization, revealing marked cell-to-cell variation in RNA abundance but relatively conserved spatial organization. cRNA was found to be predominantly nuclear, with a small but reproducible cytoplasmic fraction. Application to infected mouse lung tissue demonstrated simultaneous spatial detection of all viral RNA species. This approach provides a highly specific framework for single-cell analysis of RNA virus replication and transcription.  IMPORTANCE: Influenza virus relies on three closely related RNA molecules for replication and gene expression, yet distinguishing them inside infected cells has remained technically challenging. This limitation has hindered understanding of viral replication in space and time. We developed triple-lock fluorescence in situ hybridization, which enables simultaneous visualization of all three viral RNA species across all eight influenza genome segments in infected cells and tissues. Single-cell analysis revealed striking heterogeneity in viral RNA abundance across infected cells, whereas intracellular RNA localization patterns remained relatively conserved. Complementary RNA, a replication intermediate long thought to be strictly nuclear, was also detected at low levels in the cytoplasm. These findings provide new insight into influenza virus replication and establish a broadly applicable tool for studying RNA virus biology.
SN  - 2150-7511
ER  -