Development and application of reporter-expressing mononegaviruses: Current challenges and perspectives

Abstract

Reverse genetics allows the generation of recombinant viruses entirely from cDNA. One application of this technology is the creation of reporter-expressing viruses, which greatly increase the detail and ease with which these viruses can be studied. However, there are a number of challenges when working with reporter-expressing viruses. Both the reporter protein itself as well as the genetic manipulations within the viral genome required for expression of this reporter can result in altered biological properties of the recombinant virus, and lead to attenuation in vitro and/or in vivo. Further, instability of reporter expression and purging of the genetic information encoding for the reporter from the viral genome can be an issue. Finally, a practical challenge for in vivo studies lies in the attenuation of light signals when traversing tissues. Novel expression strategies and the continued development of brighter, red and farred shifted reporters and the increased use of bioluminescent reporters for in vivo applications promise to overcome some of these limitations in future. However, a “one size fits all” approach to the design of reporter-expressing viruses has thus far not been possible. Rather, a reporter suited to the intended application must be selected and an appropriate expression strategy and location for the reporter in the viral genome chosen. Still, attenuating effects of the reporter on viral fitness are difficult to predict and have to be carefully assessed with respect to the intended application. Despite these limitations the generation of suitable reporter-expressing viruses will become more common as technology and our understanding of the intricacies of viral gene expression and regulation improves, allowing deeper insight into virus biology both in living cells and in animals.

Specificity of Plant Rhabdovirus Cell-to-Cell Movement

“…Whether this process is mediated by nuclear export activities of a viral nucleocapsid protein or via a budding mechanism awaits future studies. With the advent of plant rhabdovirus reverse genetics systems, it is now possible to tag viral proteins genetically with fluorescent proteins, as has been shown in several mammalian rhabdovirus studies (83)(84)(85)(86)(87)(88). This approach might permit intracellular and intercellular tracking of the movement of infectious virus entities in live cells.…”

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

“…This is challenging in vitro and magnified in vivo when low numbers of infected cells are present in tissues, which must be examined using ultrathin sections. These challenges have been addressed by generating recombinant (r) viruses from clinical samples and engineering them to express fluorescent proteins from an additional transcription unit (ATU), permitting novel insights into viral pathogenesis and targeted pathological assessment in appropriate cell lines and animal models (18). To extend these studies, we obtained the genome sequence of HRSV B05 , a wildtype subgroup B strain.…”

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

“…The first replication competent influenza A reporter virus was that of Manicassamy and colleagues [ 12 ], where GFP was introduced into the NS gene segment of influenza A/PR/8/34 (H1N1) (A/PR/8). This virus represented a significant advancement in influenza virus research, as it facilitated the microscopic analysis of infected tissue [ 10 , 11 , 29 ]. Fluorescent reporter viruses, in combination with microscopy, can be used to visualise the site of virus replication and morphological changes in the cell following infection.…”