Materials and Methods: Twenty-five rats were subjected to unilateral stereotaxic injection of 10 µL LPS (1 mg/mL), while another ten rats were injected with phosphate-buffered saline (PBS, 10 µL) as control. Then, 29 parameters of rat behavior related to sickness were tracked by a device software (SMART 3.0.1) on days 0 and 14 of CN treatment. The acquired and accumulated data were analyzed using multivariate data analysis with the SIMCA Software package (version 13, Umetrics AB; Umeå, Sweden). The pattern trends of related groups were documented using PCA and OPLS analysis.

Results: A similar ameliorated correlation pattern was detected between improvement in physiological sickness behavior and anti-inflammatory biomarkers by the 1H NMR spectra of the sera following treatment with CN (500 and 1000 mg/kg body weight (bw)) and the control drug (dextromethorphan hydrobromide, 5 mg/kg of rats bw) in rats. Here, 21 biomarkers were detected for neuroinflammation. Treatment with the aqueous CN extract resulted in a statistically significant alteration in neuroinflammation metabolite biomarkers, including ethanol, choline, and acetate.

Methods: 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.

Results: 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.