The emergence of novel virus strains and the associated outbreaks are becoming a significant threat to 37 mankind (Koven 2020) . The currently ongoing pandemic, caused by the Severe Acute Respiratory 38 Syndrome-Coronavirus 2 (SARS-CoV-2), has brought the majority of the world to a grinding halt, severely 39 impacting health & economy across the nations ( fomites. Thus, the development of low cost and easily scalable antiviral coating materials, which could be 59 widely applied to various surfaces in order to inactivate the virus particles in the environment, may serve 60 as an effective way to reduce the chance of infection and hence to lower the overall speed of transmission. 61 62 Different metal oxides, including Cu and Ag have been explored for their biocidal activity in soluble as 63 well as in insoluble forms (Minoshima et al. 2016 ) . Copper and silver nanoparticles have remarkable 64 sequence from porcine teschovirus. The Nano-Luc-influenza A reporter virus, as a part of its gene 134 expression, synthesizes the PA-2A-NLuc polypeptide, which gets self-cleaved to produce Nano-luciferase. 135
Subsequently, the luciferase activity could be measured as a quantitative estimate of viral gene expression 136 and hence progression of virus replication cycle inside the cells. To test whether the Nano-luciferase activity 137 could actually serve as a proxy to virus replication, we have infected MDCK cells with different amounts 138 of input virus and viral replication/gene expression was monitored using Nano-Glo assay (Promega). As 139 shown in Figure 3A , there is a linear relationship between multiplicity of infection (0.01-0.1) and luciferase 140 light unit measurements (R 2 = 0.9294), where an increase in one log in the input virus amount leads to about 141 50% increase in the luciferase activity or vice versa, measured at 8 hours of post-infection. This data 142 suggests that the Nano-luciferase influenza A reporter virus could serve as an excellent tool to study the 143 antiviral activity of various nanoparticles or their nanocomposites used in this study. 144
In order to test the antiviral activity, we have standardized a "Nano-Luc reporter assay" described in Figure  145 3B. Briefly, Nano-luciferase influenza A reporter viruses were pre-incubated with the 5uM colloidal 146 suspensions of each of the nanoparticles/ composites or with the vehicle control for 30 minutes at room 147 temperature and subsequently used to infect MDCK cells at an MOI of 0.1. Luciferase activity was 148 measured at 8 hours of post infection and plotted as a relative percentage of the vehicle control set ( Figure  149 3C). Prior treatment of the virus stock solution with Cu-Gr composite showed 64% reduction in viral gene 150 expression, while prior treatment with Ag-Gr resulted in 20% reduction. Treatment with other materials 151 shows no significant decrease in luciferase activity. From the correlation of input virus units and the 152 corresponding luciferase activity, as shown in Figure 3A , it can be inferred that prior treatment with Cu-Gr 153 solution resulted in more than 10-fold reductions in the infectious virus population that has been used to 154 infect the MDCK cells. In this context, it should be noted that none of the materials showed substantial 155 cytotoxicity upon Madin-Darby Canine Kidney cells (MDCK) within the concentration range of 0.5 uM -156 5.0 uM as evaluated using MTT assay ( Figure 2) . Hence, the reduction in Nano-luciferase activity as a 157 result of prior exposure to Cu-Gr should be attributed exclusively to the reduction of the infectivity of the 158 Nano-luciferase reporter virus. Henceforth, we focused upon the extensive characterization of the Cu-Gr 159 nanocomposite. 160 161
We have extensively characterized the structural parameters of the synthesized Cu-Gr nanocomposites by 163 optical measurements. Figure 4 A depicts Raman spectra of synthesized Cu-Gr nanocomposite samples at 164 excitation of 532 nm in the range of 200 cm -1 to 3000 cm -1 . With Raman spectroscopy, we are able to cm -1 ) confirms the existence of graphene in the samples synthesized. Generally, D peak originates from 167 defects in the hexagonal sp 2 carbon system while the G peak arises due to the stretching vibration of sp 2 168 carbon pairs in both rings and chains (Ferrari et al. 2006 ). Except, D and G peak, the 2D peak arises at 169 ~2700 cm -1 . The 2D peak originates due to transverse optical (TO) phonons around the K point and is reporter assay in order to identify the optimal time and concentration required for its antiviral activity. The 184
Nano-Luc reporter assay was performed where the influenza A reporter virus was pretreated with the 185 colloidal form of the Cu-Gr nanocomposite for various time periods before using them for infecting MDCK 186 cells. As evidenced from Figure 5A , a sharp decrease (>50%) in the reporter activity was observed as a 187 result of 30 minutes of preincubation with Cu-Gr composite, while longer times of preincubation showed 188 only minor additional reduction. This data suggested that 30 minutes of preincubation with Cu-Gr 189 composite can lead to more than tenfold reduction in input virus titer that ultimately results in ~50% 190 decrease in reporter activity. Subsequently, we tried to identify the optimal concentration of the Cu-Gr 191 composite required for its antiviral activity. Different concentrations of the Cu-Gr composite (50nM, 192 100nM, 500nM, 1µM, 2µM and 5µM, respectively) were used to treat the Nano-Luc influenza A reporter 193 virus for 30 minutes followed by performing Nano-Luc reporter assay with the same. 1µM Cu-Gr nanocomposite shows non-significant decrease in viral titer. This data further substantiates the 207 fact that treatment with 5µM Cu-Gr significantly reduces viral infectivity which results in a decrease in 208 viral gene expression, replication and subsequent production of viral titer ( Figure 5C ). The plaque assay 209 titer data are tabulated in Figure 5D . Each data is a representative of at least three independent experiments, each experiment was performed in 386 triplicate. Graphs are performed in Microsoft Excel and represented as mean standard deviations (n=3). 387
Results were compared by performing two-tailed Student's t test. Significance is defined as p<0.05 and 388 statistical significance is indicated with an asterisk (*). The *p value < 0.05, **p value < 0.01 **p value 389 and ***p < 0.001 were considered statistically significant. 