Cell Cultures for Virology: Usability, Advantages, and Prospects

Abstract

Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.

Keywords: cell cultures; cell permissivity; cell susceptibility; cell-based method; reporter construction; virus; virus-inducible expression.

Figure 1

Molecular reporter system for ssRNA (−) genome virus detection. The principle of the reporter system is based on the production of mRNA without the cap and the polyA tail. Such RNAs mimic the virus genome. Ribozymes hydrolyze RNA or RNA-polymerase I promoters. This transcript mimics (−) viral RNA, which allows it to be replicated using viral proteins. The transcription of the reporter protein occurs from (+) RNA obtained in this manner.

Figure 2

Molecular reporter system for ssRNA (+) genome viruses with subgenomic RNA detection. The reporter system scheme is based on the capability of such viruses to synthesize structural proteins from subgenomic (+) RNA as a product of virus replication. In the first stage, mRNA mimicking the virus (+) RNA will be produced in the cell. During infection, the (+) RNA is recognized by viral proteins and replicates to form the (−) RNA copy. In the next step, this (−) RNA serves as a matrix strand for the transcription of subgenomic (+) RNA; the late promoter is activated by viral proteins, and the reporter protein can be synthesized.

Figure 3

Molecular reporter system for ssRNA (+) genome viruses with genomic polyprotein detection. The reporter system is based on proteolytic cleavage by a specific viral protease in the processing of the primary viral polypeptide to generate the final proteins. The reporter protein binds to the viral protein at the cleavage sites of the viral protease and is released after infection.

Figure 4

Molecular reporter system mimicking the genome for Hepatitis C virus (HCV) detection. After the transcription of the mRNA, which carries all the elements of the reporter construct, the terminal fragments are self-pinched off by ribozymes. Such mRNAs mimic the (−) RNA viral genome. Viral proteins recognize this RNA and replicate it during infection. On the (+) RNA chain, there is an internal ribosome entry site for reporter protein synthesis.

Figure 5

Molecular reporter system for retrovirus and dsDNA genome virus detection. In noninfected cells, there is no mRNA synthesis from the viral promoter. After the infection of the cell occurs, the transcription and translation of the reporter protein begin in the presence of the viral protein.