ANTIVIRAL EFFECT OF SELECTED PLANTS’ LEAF EXTRACTS AGAINST VELOGENIC STRAIN OF NEWCASTLE DISEASE VIRUS

Abstract

Newcastle Disease Virus (NDV) is a paramyxovirus, which causes Newcastle Disease (ND) in birds and conjunctivitis in humans. This viral disease is currently prevented by vaccination. However, there are challenges faced such as cost, availability, storage, accessibility of the vaccines, therefore, the need for alternative measures. In view of this, Investigation was made into the antiviral effect of methanol, n-Hexane, and aqueous leaf extracts of Phyllanthus amarus Thonn, Vernonia amygdalina Delile, and Jatropha curcas Linn against NDV. Nine-day-old viable embryonated chicken eggs (ECE) were used and were divided into nine groups. Methanol, aqueous, and n-Hexane leaf extracts of the plants were administered to the various groups through the allantoic route at varying final concentrations of 50, 25, 10, and 5 mg/mL, respectively. Group 1 was infected with 100 EID50/0.1 mL NDV kudu strain before administration of the leaf extracts, group 2 was infected with 100 EID50/0.1 mL NDV kudu strain after administration of the leaf extracts, while group 3 was infected with 100 EID50/0.1 mL NDV kudu strain at the same time the leaf extracts were administered. Negative control group received only PBS and un-inoculated control group were not inoculated, the positive control group was infected with 100 EID50/0.1 mL NDV Kudu strain only. The embryonated chicken eggs were incubated at 37oc and embryonic survival was monitored daily by candling. Allantoic fluids from eggs in the different groups were harvested after 96 hours for spot hemagglutination and hemagglutination (HA) assays. The most potent plant extract from the in-ovo assay was used for further in-vivo research. One hundred and twenty (120) day-old broiler chickens were purchased and raised for the in-vivo experiment. At three weeks, the chickens were randomly assigned into 12 groups of 10 chickens each. Chickens in groups 1, 2, 3 and 4 were vaccinated with NDV La sota vaccine while those in 5, 6, 7 and 8 were left unvaccinated. Groups 9 and 10 served as the positive controls and was infected with 100 EID50/0.1 mL NDV Kudu strain while 11 and 12 were the negative controls. To study the prophylactic effects of the extract on the virus, chickens in groups 1 and 3 were administered 250 mg/L of the leaf extracts while groups 2 and 4 were administered 500 mg/L of leaf extracts fourteen days before experimental infection. Groups 5 and 7 were administered 250 mg/L while groups 6 and 8 were administered 500 mg/L of leaf extracts for fourteen days after infection, in order to study the therapeutic effect of the leaf extracts. All groups except the negative control were infected with 100 EID50/0.1 mL NDV. Clinical signs, body weight changes, and mortality rates were documented. Antibody titres against the virus were determined and post-mortem examination was conducted. Haematology and biochemical assessment of blood samples from experimental chickens was conducted. Phytochemistry and determination of bioactive component of extract was also conducted. The in-ovo assay revealed that at the concentration of 50 mg/mL, the n-hexane leaf extract of P. amarus significantly (p<0.05) reduced viral titre from log2 10 to log2 2, while the extracts of V. amygdalina and J. curcas did not show a statistically significant reduction (p<0.05). The in-vivo analysis showed reduction (p<0.05) in mortality rates following administration of n-Hexane extract after the viral challenge. Similarly, antibody titres decreased significantly (p< 0.05) in a dosage-dependent pattern in the prophylactic group. The erythrocyte response in the chickens had varying patterns; there were significant reduction in values of total erythrocyte count, packed cell volume, and haemoglobin concentration in infected chickens. Monocytes were reduced significantly in the infected birds to 7±0.6% compared to uninfected birds which had 10±0.9%. There were no significant changes in the eosinophil and basophil absolute values in both infected and uninfected birds. The biochemical assay revealed no significant differences in the liver enzyme markers of the infected chickens but a significant reduction in the total protein content of the infected chickens. Post-mortem examination revealed petechial haemorrhages in the proventriculus. The phytochemical assay of plant leaf extracts revealed the presence of phytochemicals including alkaloids, tannins, saponins, flavonoids, phenols, steroids, and glycosides, while Gas Chromatography Mass Spectrometry of the leaf extract revealed the presence of bioactive compounds including phytol, phenol, and tocopherol. It was observed that n-hexane leaf extract of Phyllanthus amarus’ showed the highest antiviral activities against NDV and that administration of the leaf extract before infection was more effective in reducing the mortality rates of chickens due to the virus infection compared to administration after infection. This study also demonstrates that n-Hexane leaf extract of P. amarus does not show a significant increase or decrease on the haematological and selected biochemical profile of the chickens. However, NDV infection resulted in the increase in lymphocyte count of infected broiler chickens. These findings indicate that n-Hexane leaf extract of P. amarus possesses biologically active compounds which have significant antiviral activity against NDV in broiler chickens and that administration of the extract at a dosage of 500 mg/L might be a safer approach in utilization of the leaf extract against NDV