## 2 Materials and Method
### Investigations of Antiviral Activity  
To test the antiviral activities of the synthesized nanostructured particles, a standard three-step procedure was followed. These steps included inoculation, harvesting of the chorioallantoic fluid, and the Hemagglutination Assay protocol. In this procedure, a volume of 100 uL each of synthesized gold and gold-PVP colloids was separately mixed with an equal volume of confirmed Avian Influenza Virus (AIV) (H9N2) of 10\({}^{6}\) EID50 in a microfuge tube. The AIV (H9N2) of 10\({}^{6}\) EID50 was procured from Influenza lab, Institute of Microbiology, the University of Veterinary and Animal Sciences, Lahore. The mixture of nanostructured gold particles (Au-20) and gold-PVP nanostructured composites (Au-PVP-20) and the virus was separately incubated for 60 min at room temperature to interact. Inoculation is proceeded by injecting the above samples into the allantoic cavity of 9-11 days' serum pathogen-free fertile chicken eggs with a 22 G needle attached to a 1 mL syringe as shown in Figure 2a. The hole was sealed with wax. After 48 hrs. of incubation at 37 \({}^{\circ}\)C, the eggs were chilled for at least 4 h at 4 \({}^{\circ}\)C to let the virus propagate. Figure 1: (**a**) 1.0 mM (HAuCl4 3H2O); 5 mM D-Fructose precursor; (**b**) Electrochemically reduced gold colloids after 20 min of microplasma processing. Chilled eggs with air sacs facing up into a holder were transferred to the biosafety lab. The eggshell above the air sac was removed carefully with sterilized scissors without damaging the chorioallantoic membrane, as shown in Figure 2b. With sterile forceps, the chorioallantoic membrane was removed, and allantoic fluid was collected as much as possible with a small spatula or 1 mL Eppendorf pipette. The chorioallantoic fluid (CAF) harvested from the Embryonated chicken eggs as a virus stock was the major part of the laboratory-based study of influenza A virus titration in the hemagglutination Assay protocol procedure referenced by [PERSON] and [PERSON] (1977) is described below. Hemagglutination Test (HA)  
In a typical Hemagglutination assay (HA) protocol, an influenza virus titer can be evaluated by utilizing serial dilutions of a virus on a 96-well plate (either V- or round-bottom) and introducing a fixed amount of red blood cells, for example, 1% Chicken RBCs. Using a multi-channel micropipette, 50 \(\upmu\)L of normal saline (NS) was dispensed into all of the wells of the micro-titration plate. A volume of 50 \(\upmu\)L of harvested CAF was dispensed in the first well. Two-fold serial dilutions were made from 1:2 to 1:2048. It was accomplished by putting 50 \(\upmu\)L into the next well and so on until the 11 th well was reached. A total of 50 \(\upmu\)L of 1% RBC suspension was dispensed into all of the wells. The 12 th well, which contained only 1% RBC suspension and normal saline, was preserved as a negative control. The micro-titration plate was then incubated for 20-30 min at 37 \({}^{\circ}\)C. The RBCs in the 12 th well had settled down to form a visible bead at the bottom of the well. To assure quality findings, the HA test was repeated three times. HA activity negative means that the virus has been disabled or have become neutral with activity of nanoparticles. Consequently, RBCs have settled down to the bottom of the well in the form of a bead. HA activity positive confirms the presence of a virus that adheres to the surface of RBCs causing agglutination. Consequently, preventing RBCs from settling down and a matt reddish suspension is seen. The HA activity was recorded as shown in Figure 3. The Figure 3a shows HA activity of Au-20 and dilution, upto which their tendency of inhibiting influenza virus activity of agglutination is observed. Whereas in Figure 3b, the dilution of Au-PVP-20 upto which their ability of inhibiting virus activity of agglutination is displayed. The virus has a tendency to adhere to the surface of red blood cells, causing agglutination and hence preventing them to settle at the bottom. This is called HA activity positive.