Nuphar lutea Extracts Exhibit Anti-Viral Activity against the Measles Virus

Abstract

Different parts of Nuphar lutea L. (yellow water lily) have been used to treat several inflammatory and pathogen-related diseases. It has shown that Nuphar lutea extracts (NUP) are active against various pathogens including bacteria, fungi, and leishmanial parasites. In an effort to detect novel therapeutic agents against negative-stranded RNA (- RNA) viruses, we have tested the effect of a partially-purified alkaloid mixture of Nuphar lutea leaves on the measles virus (MV). The MV vaccine's Edmonston strain was used to acutely or persistently infect cells. The levels of several MV proteins were detected by a Western blot and immunocytochemistry. Viral RNAs were quantitated by qRT-PCR. Virus infectivity was monitored by infecting African green monkey kidney VERO cells' monolayers. We showed that NUP protected cells from acute infection. Decreases in the MV P-, N-, and V-proteins were observed in persistently infected cells and the amount of infective virus released was reduced as compared to untreated cells. By examining viral RNAs, we suggest that NUP acts at the post-transcriptional level. We conclude, as a proof of concept, that NUP has anti-viral therapeutic activity against the MV. Future studies will determine the mechanism of action and the effect of NUP on other related viruses.

Keywords: Nuphar lutea plant extract; anti-viral natural products; herbal medicines; measles virus; nupharidines.

Figure 1

The effect of Nuphar lutea (NUP) on survival of measles virus (MV) acutely infected cells. VERO cells were infected with double dilutions of measles virus (MV) (maximum Multiplicity of Infection (MOI)—2.5). The cells were treated with 0.3 µg/mL NUP, either 24 h before infection (dots), simultaneously (light grey), or 24 h after infection (stripes). Cell survival was determined by the tetrazolium based cell proliferation (viability) test 96 h post infection. Statistical analysis (p < 0.05 *, p < 0.01 **) and a moving average trend line is presented. MV infected but Nuphar lutea (NUP) untreated cells were used as a reference for statistical analysis.

Figure 2

L428 cells persistently infected with MV-GFP. The GFP MV virus was detected by fluorescence microscopy (X 200).

Figure 3

Expression of P- and N-MV proteins following NUP treatment. L428 + MV cells were incubated for 12 h without NUP (Control) or with 6 µg/mL NUP. Immunohistochemistry with antibodies against P- and N-MV proteins was performed (X 400). Positive staining is brown. The nuclei were counterstained with hematoxylin (blue).

Figure 4

Expression of P-, N-, and V-MV proteins following NUP treatment by a Western blot. Western blot analysis was performed on whole cell lysates from L428 + MV cells. Antibodies used were against P-, N-, and V-MV proteins. L428 + MV cells were incubated with different concentrations of NUP for 12 h. Densitometry of the blot is presented and normalized to β-actin.

Figure 5

Effect of NUP on P- and N-MV genes’ RNA expression. L428 + MV cells were incubated with different concentrations of NUP for 12 h. After incubation, total RNA isolation and qRT-PCR were performed. MV primers and probes were against N- and P- protein, human β-actin, and PHP genes were used for normalization. After normalization of N- or P- cycles with either β-actin or PHP, the control values were subtracted from the values of the normalized NUP-treated samples (∆Ct). (A) N gene normalized to β-actin gene, (B) P- gene normalized to β-actin gene, (C) N- gene normalized to PHP gene, and (D) P-gene normalized to PHP gene.

Figure 6

Members of the thioalkaloid family present in the NUP semi-purified extract.