**2.17. Evaluation for antiviral activity**

Three experiments were conducted.

## *2.17.1. Experiment 1*

One hundred and five emberyonated chicken eggs (ECEs) were examined; equal volumes of HPAI H5N1 virus and original extracts were separately used at three levels:

*Level 1:* Equal volumes of HPAI H5N1 virus and the original undiluted samples were mixed and incubated at room temperature for 1 h then inoculated into the allantoic sac of five ECEs for each product sample at dose 0.2 mL/ECE.

*Level 2:* Equal volumes of HPAI H5N1 virus and the 1/5 dilution of each sample were mixed and preceded as level 1.

*Level 3:* Equal volumes of the virus and the 1/10 dilution of each sample were mixed and preceded as level 1. In addition, five ECEs were inoculated with the virus that mixed with equal volume of saline at a dose of 0.2 mL/ECE of saline alone (negative control). The ECEs are inoculated at 37 °C and candled every 2 h till all the positive control ECEs died.

## *2.17.2. Experiment 2*

200 The Complex World of Polysaccharides

**2.15. Cell viability assay** 

under the phase contrast microscope.

**2.16. Haemagglutinating activity assay** 

**2.17. Evaluation for antiviral activity** 

for each product sample at dose 0.2 mL/ECE.

Three experiments were conducted.

*2.17.1. Experiment 1* 

and preceded as level 1.

technique of haemagglutination (HA) test (Takatsy, 1955).

**2.14. Cell morphology evaluation by inverted light microscopy** 

Morphological changes were scored (Simoes et al., 1999).

Hep-2 cell cultures (2× 105 cells/mL) were prepared in 96-well tissue culture plates (Greiner-Bio one, Germany). After 24 h incubation at 37 °C in a humidified 5% (v/v) CO2 atmosphere cell monolayers were confluent, the medium was removed from each well and replenished with 100 \_L of bi-fold dilutions of different samples tested prepared in DMEM (GIBCO BRL). For cell controls 100 \_L of DMEM without samples was added. All cultures were incubated at 37 °C in a humidified 5% (v/v) CO2 atmosphere for 72 h. Cell morphology was observed daily for microscopically detectable morphological alterations, such as loss of confluence, cell rounding and shrinking, and cytoplasm granulation and vacuolization.

It was done by trypan blue dye exclusion method (Walum et al., 1990). Hep-2 cell cultures (2×105 cells/mL) were grown in 12-well tissue culture plates (Greiner-Bio one, Germany). After 24 h incubation, the same assay described above for tested samples cytotoxicity was followed by applying 100 L of tested samples dilutions (bifold dilutions) per well. After 72 h the medium was removed, cells were trypsinized and an equal volume of 0.4% (w/v). Trypan blue dye aqueous solution was added to cell suspension. Viable cells were counted

This was applied for the allantoic fluids of the inoculated eggs and measured by micro

One hundred and five emberyonated chicken eggs (ECEs) were examined; equal volumes of

*Level 1:* Equal volumes of HPAI H5N1 virus and the original undiluted samples were mixed and incubated at room temperature for 1 h then inoculated into the allantoic sac of five ECEs

*Level 2:* Equal volumes of HPAI H5N1 virus and the 1/5 dilution of each sample were mixed

*Level 3:* Equal volumes of the virus and the 1/10 dilution of each sample were mixed and preceded as level 1. In addition, five ECEs were inoculated with the virus that mixed with equal volume of saline at a dose of 0.2 mL/ECE of saline alone (negative control). The ECEs

are inoculated at 37 °C and candled every 2 h till all the positive control ECEs died.

HPAI H5N1 virus and original extracts were separately used at three levels:

One hundred and five SPF ECEs were used in this experiment; equal volumes of HPAI H5N1 virus and the original samples were mixed with equal volume of the original samples and inoculated directly into the allontoic sac of five ECEs for each product sample at a dose of 0.20 mL/ECE for each product sample at a dose of 0.2 mL/ECE. Five ECEs were inoculated with equal volume of the HPAI H5N1 virus and saline at dose of 0.2 mL/ECE (positive control). Another five ECEs were inoculated with 0.20 mL/ECE of saline alone (negative control). All the ECEs were incubated at 37 °C and controlled every 2 h till the ECEs of the positive control died

## *2.17.3. Experiments 3*

One hundred and five SPF ECEs of nine days old were used in this experiment. 0.10 mL of the HPAI H5N1 virus was inoculated via the allontoic sac of each ECE into 100 ECEs and then the inoculated ECEs were incubated for 1 h at 37 *°*C. The original samples were inoculated into five ECEs, which previously inoculated with the virus at a dose of 0.1 ml. Another five ECEs were inoculated with 0.2 ml/ECE of the mixed virus and saline. Other five ECEs were inoculated with 0.2 ml/ECE of saline alone. The ECEs were inoculated at 37 °C and candled every 2 h till ECEs of the positive control died.

## **2.18. Antiviral effect of tested samples on adenovirus type 40**

Seventy five microliters of non toxic dilutions were mixed with 75 L of different doses 1 × 104, 1 × 105, and 1 × 106 infectious viral particles/mL of adenovirus type 40 provided by American Type Culture Collection (ATCC). Then the mixture was incubated overnight at 4 °C. Inoculation of 100 L of 10 fold dilutions of treated and untreated adenovirus was done into Hep-2 cell line in 12 multi well-plates. After 1 h incubation for adsorption at 37 °C, 1 mL medium (DMEM) was added to each well. The cell line was observed daily for one week then, three times freezing and thawing for tested plates were done. Nested PCR was done for confirmation of adenovirus (presence/absence) in each well (Puig et al., 1994).
