Abstract
A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.
Highlights
Human norovirus (HuNV) is one of the leading causes of non-bacterial acute gastroenteritis worldwide and is responsible for a large number of epidemics annually [1]
We developed a NanoLucTM Luciferase-tagged replicon system to study murine norovirus (MNV)-1
The first part focused on the designing and construction of an Murine Norovirus-1 (MNV-1) based NanoLucTM Luciferase-tagged replicon, whereas the second part had to do with the cloning of the constructed reporter gene into MNV-1 cDNA clones
Summary
Human norovirus (HuNV) is one of the leading causes of non-bacterial acute gastroenteritis worldwide and is responsible for a large number of epidemics annually [1]. The clinical manifestations of HuNV infection include acute and rapidonset diarrhea, nausea, vomiting, headache, fever, anorexia and malaise. Aside from these classical symptoms, the infection can lead to severe clinical outcomes, such as necrotising enterocolitis, seizures in infants, encephalopathy, pneumatosis intestinalis and disseminated intravascular coagulation [3,4,5,6,7,8,9]. Many of the biological functions and properties of HuNV are not properly understood due to the lack of an efficient cell culture system or a smallanimal model that may be used to study human norovirus [13]. The NanoLucTM Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication
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