Determination of virus titer VERO cells of 1 × 10 5 cells/mL were applied 100 µL per well to 96-well microtiter plates and cultured for 24 hours. After discarding medium, 100 µL of infected supernatant, which was 10 −1 to 10 −10 diluted with medium, was added to each well (quadruplicate) until CPE did not increase. TCID 50 (50 percent tissue culture infective dose) was calculated according to Reed and Muench. 19, 20 Measurement of cytotoxicity The cytotoxicity of PEGylated α-MMC and PEGylated MAP30 was evaluated by the quantitative MTT test. 21 A total of 1 × 10 5 VERO cells/mL was applied to 96-well microtiter plates at 100 µL per well and was exposed to un-PEGylated proteins and PEGylated-proteins at 3.3 µmol ⋅ L −1 ∼3.3 nmol ⋅ L −1 (100 mg ⋅ L −1 ∼0.1 mg ⋅ L −1 ) and acyclovir (ACV) at 10 µmol ⋅ L −1 ∼0.01 µmol ⋅ L −1 for 48 hours as well as cells without drugs as control. Each concentration was tested in quadruplicate. The determination procedure was carried out according to the previous description. 18 The OD at 490 nm was measured using ELISA: