Antiviral activity of hemolymph of Podalia against rubella virus

Abstract

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology. Rubella virus (genus Rubivirus, family Togaviridae) is a single stranded RNA virus of positive genome polarity. Rubella virus infection of susceptible women during the first trimester of pregnancy often results in long-term virus persistence in the fetus causing multiple organ abnormalities. Potent antiviral activity against rubella virus (RV) has been observed in the hemolymph of Podalia sp. (Lepidoptera: Megalopygidae). This study evaluated the effect of hemolymph on RV infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Results of cell viability and cell proliferation assays indicated that hemolymph was not toxic to cultured SIRC cells. Viral binding assay, antiviral assay, PCR, real-time PCR, and transmission electron microscopy were used to demonstrate that hemolymph in post-treatment could inhibit the production of infectious RV particles. Specifically, hemolymph was found to inhibit RV adsorption to the SIRC cells.

Keywords: Antiviral; Congenital Rubella Syndrome; Hemolymph; Rubella virus.

Fig. 1

Cell viability of SIRC cells treated with different concentrations of hemolymph. The number represent the mean of three replicates

Fig. 2

Inhibitory effect of hemolymph on rubella viruses RNA synthesis in SIRC cells as analyzed by qRT-PCR. Cell culture lysates were collected after pre-treatment and post-treatment with different concentrations from hemolymph (32, 68, 88, 136 and 180 mg/mL) for 72 h. The inhibitory effect was determined using qPCR. The infectivity from RV decrease after post treatment with hemolymph. The error bars represent the SD calculated from three independent experiments in triplicate. Note the post-treatment the % inhibitory RV infection was more 90%

Fig. 3

Binding assay of untreated RV or RV treated with hemolymph (68, 136 and 200 mg/mL). Infectivity was determined by qPCR. The numbers represent the mean triplicate

Fig. 4

Agarose gel of products obtained by conventional RT-PCR of representative RV glycoprotein E1. Products obtained by RT-PCR for the 185-bp amplicon. The samples in each lane are as follows: Lane 1 DNA ladder, with visible bands identified to the left in base pairs (bp). Lane 2 SIRC cells inoculated with RV (DNA amplified with primers for RV). The lanes 3, 4 and 5 cells treated with 68, 136 and 200 mg/mL of hemolymph. Lane 6 negative sample

Fig. 5

SIRC cells were cultivated on Aclar film and after 48 hrs were treated or untreated with hemlymph and infected with RV were processed by TEM. a–c SIRC cells inoculated with RV treated with hemolymph. Note the Golgi system (GC), Mitochondria (M). Note that in SIRC cells infected with RV and treated with hemolymph no replication complex or viral particles were found. c SIRC cells morphology (elongated fibroblaste-like cells). d–f. SIRC cells inoculated with RV but untreated with hemolymph. Note the replication complex (CPV) and RV like particles. f SIRC cells showed cytophatic effect (CPE)