Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been detected in human breastmilk, infants' safety with breastmilk feeding is of great concern for women with coronavirus disease 2019 (COVID-19). 1 It is known that milk has antiviral properties. 2 However, little is known about the antiviral property of human breastmilk to SARS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that whey protein from human breastmilk effectively inhibited both SARS-CoV-2 and GX_P2V by blocking viral attachment and viral replication at entry and even post entry. Moreover, human whey protein inhibited infectious virus production, as proved by the plaque assay. We found that whey protein from different species, such as cow and goat, also showed anti-coronavirus properties. Commercial bovine formula milk also showed similar anti-SARS-CoV-2 activity. Firstly, healthy human breastmilk samples collected in 2017 and stored properly at −80°C were tested for their potential effects on SARS-CoV-2 infection. Mothers provided informed consent. This study was approved by the ethics committees of the Medical Center and all samples were anonymized. The skimmed breastmilk was obtained after removal of the lipid fraction. Vero E6 cells were infected with a mixture of SARS-CoV-2 pseudovirus (650 TCID 50 /well) and human breastmilk (4 mg/ ml). Human breastmilk from eight donors showed a significant inhibition of more than 98% of the SARS-CoV-2 pseudovirus. As reported recently, a SARS-CoV-2-related pangolin coronavirus model (GX_P2V) 3 shares 92.2% amino acid identity in spike protein with SARS-CoV-2, which is a suitable model for SARS-CoV-2 infection research. We utilized GX_P2V (MOI: 0.01 in Vero E6 cells) as the model to study the effect of breastmilk on viral infection and also found similar results (Fig. 1a). The inhibition is concentration dependent with an EC 50 of 0.13 mg/ml of total protein (Fig. 1b and Supplementary Fig. S1) in the SARS-CoV-2 pseudovirus model. Consistent with the SARS-CoV-2 study, the GX_P2V model also showed inhibition with an EC 50 of about 0.5 mg/ml of total protein (Fig. 1c and Supplementary Fig. S2). Interestingly, human breastmilk did not show any cytotoxicity to Vero E6 cells (CC 50 > 3 mg/ml), and even promoted cell proliferation. These results indicated that human breastmilk showed high anti-SARS-CoV-2 and anti-GX_P2V property, but limited cytotoxicity to Vero E6 cells. We then assessed the impact of human breastmilk on infectious virus production in Vero E6. RT-qPCR analysis of the GX_P2V virus from supernatant showed that even 0.16 mg/ml of breastmilk significantly blocked viral production (Fig. 1d). Western blot of viral nucleoprotein also showed similar results (Fig. 1e). To investigate the infectious virus, we performed plaque assay. As shown in Fig. 1f, the plaque assays showed that live viruses were significantly lower in breastmilk treatment compared to the control group, which confirmed that

Since the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human breastmilk, little is known about the antiviral property of human breastmilk to SARS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that whey protein from human breastmilk effectively inhibited both SARS-CoV-2 and GX_P2V by blocking viral attachment, entry and even post-entry viral replication. Moreover, human whey protein inhibited infectious virus production proved by the plaque assay. We found that whey protein from different species such as cow and goat also showed anti-coronavirus properties. And commercial bovine milk also showed similar activity. Interestingly, the main antimicrobial components of breastmilk, such as Lactoferrin and IgA antibody, showed limited anti-coronavirus activity, indicating that other factors of breastmilk may play the important anti-coronavirus role. Taken together, we reported that whey protein inhibits SARS-CoV-2 and its related virus of GX_P2V. These results rule out whey protein as a direct-acting inhibitor of SARS-CoV-2 and GX_P2V infection and replication and further investigation of its molecular mechanism of action in the context of COVID-19.

Since the first reported case caused by the novel coronavirus SARS-CoV-2 infection in Wuhan, COVID-19 has caused serious deaths and an ongoing global pandemic, and it is still raging in more than 200 countries and regions around the world and many new variants have appeared in the process of continuous transmission. In the early stage of the epidemic prevention and control and clinical treatment, traditional Chinese medicine played a huge role in China. Here, we screened out six monomer compounds, including artemether, artesunate, arteannuin B, echinatin, licochalcone B and andrographolide, with excellent anti-SARS-CoV-2 and anti-GX_P2V activity from Anti-COVID-19 Traditional Chinese Medicine Compound Library containing 389 monomer compounds extracted from traditional Chinese medicine prescriptions “three formulas and three drugs”. Our discovery preliminary proved the stage of action of those compounds against SARS-CoV-2 and provided inspiration for further research and clinical applications.

SARS-CoV-2, which likely spilled over from wildlife, is the third highly pathogenic human coronavirus. Being highly transmissible, it is perpetuating a pandemic and continuously posing a severe threat to global public health.

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