Non-steroidal anti-inflammatory drugs play a vital role due to their multi therapeutic approach. In this study, the antiviral activity of indomethacin, naproxen, and mefenamic acid against avian influenza H9 virus was evaluated In ovo. The stock solutions of each drug were prepared in their perspective solvent and preserved. From the stock, three different dilutions (10µg/ml, 20µg/ml, 40µg/ml of indomethacin, 25µg/ml, 50µg/ml, 100µg/ml of naproxen and 20µg/ml, 40µg/ml, 80µg/ml of mefenamic acid) of each drug were prepared. For each of drug to be tested, 25 embryonated chicken eggs were assigned to 5 groups having 5 eggs each, to evaluate both antiviral activity and embryonic toxicity parameters.
For evaluating antiviral activity, the groups of embryonated chicken eggs were inoculated with 4HA virus, antibiotics and different concentrations of indomethacin, naproxen and mefenamic acid. For evaluation of embryonic toxicity, embryos of each group were injected with normal saline, antibiotics and different concentrations of indomethacin, naproxen and mefenamic acid. Two controls i.e. positive control of virus (received 4HA Virus only) and negative control (received normal saline) were also included to validate the test results.
With avian influenza H9 virus the different concentrations of each drug were mixed and 0.2 ml of this suspension was inoculated to 9th to 10th day embryonated eggs along with positive and negative controls having only virus and normal saline respectively. Amantadine, standard drug, was inoculated by following the mentioned manner. These inoculated embryonated chicken eggs were incubated at 37oC and were checked after 12 – 72 hours. After 72 hours of post inoculation, chilling was done by placing all the eggs at 4oC in fridge for overnight section of time and the allantoic fluid was collected.
The embryo survival percentage, positive or negative spot haemagglutination activity and determination of virus titre by haemagglutination test confirmed the antiviral activity. The embryonic toxicity effects of indomethacin, naproxen, mefenamic acid and amantadine were assessed by only inoculating the drug of respective concentrations as used for antiviral activity in embryonated chicken eggs and incubating for 72 hours. Among the three non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin showed significant antiviral activity against influenza H9 virus as compared to naproxen and mefenamic acid. Naproxen showed antiviral activity against influenza H9 virus greater than that of mefenamic acid. However, antiviral activity of mefenamic acid as compared to naproxen and indomethacin is negligible against influenza H9 virus when confirmed by Spot Hemagglutination test while reduction in viral titre was observed by Hemagglutination test.