Establishment of a genetically engineered chicken DF-1 cell line for efficient amplification of influenza viruses in the absence of trypsin

Kelly Chungu,Seung Je Woo,Jae Yong Han,Young Hyun Park,Hong Jo Lee,Deivendran Rengaraj,Su Bin Lee

doi:10.1186/s12896-020-00663-6

Abstract

BackgroundThe initial step of influenza infection is binding of the virus to specific sialic acid receptors expressed by host cells. This is followed by cell entry via endocytosis. Cleavage of the influenza virus hemagglutinin (HA) protein is critical for infection; this is performed by host cell proteases during viral replication. In cell culture systems, HA is cleaved by trypsin added to the culture medium. The vast majority of established cell lines are mammalian.ResultsIn the present study, we generated genetically engineered chicken DF-1 cell lines overexpressing transmembrane protease, serine 2 (TMPRSS2, which cleaves HA), ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1, which plays a role in synthesis of α-2,3 linked sialic acids to which avian-adapted viruses bind preferentially), or both. We found that overexpression of TMPRSS2 supports the virus life cycle by cleaving HA. Furthermore, we found that overexpression of ST3GAL1 increased the viral titer. Finally, we showed that overexpression of both TMPRSS2 and ST3GAL1 increased the final viral titer due to enhanced support of viral replication and prolonged viability of the cells. In addition, overexpression of these genes of interest had no effect on cell proliferation and viability.ConclusionsTaken together, the results indicate that these engineered cells could be used as a cell-based system to propagate influenza virus efficiently in the absence of trypsin. Further studies on influenza virus interactions with chicken cell host factors could be studied without the effect of trypsin on cells.

Highlights

The initial step of influenza infection is binding of the virus to specific sialic acid receptors expressed by host cells
Establishment of Transmembrane serine protease 2 (TMPRSS2)- and Transmembrane serine protease 4 (TMPRSS4)-overexpressing cell lines and subsequent viral challenge The presence of trypsin-like proteases that cleave HA means that influenza viruses preferentially infect the respiratory and gastrointestinal tracts
We compared the distribution of TMPRSS2 and TMPRSS4 in lung, trachea, liver, small intestine, and large intestine samples from White Leghorn (WL) chickens aged 18 weeks and wild-type (WT) DF-1 cells by quantitative real-time PCR

Summary

Introduction

The initial step of influenza infection is binding of the virus to specific sialic acid receptors expressed by host cells. This is followed by cell entry via endocytosis. Cleavage of the influenza virus hemagglutinin (HA) protein is critical for infection; this is performed by host cell proteases during viral replication. The influenza virus surface protein hemagglutinin (HA) plays two major roles during the early life cycle of the virus: it binds to cell surface receptors and facilitates fusion of viral and endosomal membranes to release viral RNA (vRNA) into the cytoplasm [1]. Recent studies show that Madin-Darby canine kidney (MDCK) cell lines expressing proteolytic enzymes such as TMPRSS2, HAT, and Mosaic serine protease large form cleave HA in the absence of trypsin [1, 11]

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