key: cord-1048536-5t9fo6lp
authors: Joseph, Jeswin; T, Karthika; Ajay, Ariya; Das, V.R. Akshay; Raj, V. Stalin
title: Green tea and Spirulina extracts inhibit SARS, MERS, and SARS-2 spike pseudotyped virus entry in vitro
date: 2020-06-23
journal: bioRxiv
DOI: 10.1101/2020.06.20.162701
sha: 660b5bf5983144f337a110ff97d3d7b2b52f76b8
doc_id: 1048536
cord_uid: 5t9fo6lp

Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike (S) protein to bind to a host cell surface receptor. The rapid outbreak of emerging novel coronavirus, SARS-CoV 2 in China inculcates the need for the development of hasty and effective intervention strategies. Medicinal plants and natural compounds have been traditionally used to treat viral infections. Here, we generated VSV based pseudotyped viruses (pvs) of SARS-, MERS-, and SARS-2 CoVs to screen entry inhibitors from natural products. In the first series of experiments, we demonstrated that pseudotyped viruses specifically bind on their receptors and enter into the cells. SARS and MERS polyclonal antibodies neutralize SARSpv and SARS-2pv, and MERSpv respectively. Incubation of soluble ACE2 inhibited entry of SARS and SARS-2 pvs but not MERSpv. In addition, expression of ACE2 and DPP4 in non-permissive BHK21 cells enabled infection by SARSpv, SARS-2pv, and MERSpv respectively. Next, we showed the antiviral properties of known enveloped virus entry inhibitors, Spirulina and Green tea extracts against CoVpvs. SARSpv, MERSpv, and SARS-2pv entry were blocked with higher efficiency when preincubated with either green tea or spirulina extracts. Green tea provided a better inhibitory effect than the spirulina extracts by binding to the S1 domain of spike and blocking the interaction of spike with its receptor. Further studies are required to understand the exact mechanism of viral inhibition. In summary, we demonstrate that pseudotyped virus is an ideal tool for screening viral entry inhibitors. Moreover, spirulina and green tea could be promising antiviral agents against emerging viruses.

The past three decades have witnessed a tremendous increase in the number of highly pathogenic 44 emerging viruses [1] which have caused serious global threats initiating diverse approaches for 45 understanding the biology of the virus and for robust vaccine and therapeutic development. Cold water extraction of Spirulina (Arthrospira platensis) and Green Tea. Spirulina and green 182 tea are available in routine organic/medical stores. Spirulina was purchased as capsules or powder 183 and green tea was purchased as dried leaves. Extracts were prepared as described elsewhere [33] . 184

Briefly, Spirulina and green tea were powdered using mortar and pestle and the fine powder was 185 weighed at 20mg/ml concentration and were mixed in sterile distilled water and vortexed for 5 186 min. Next, the mixtures were freeze-thawed and the supernatants were collected by centrifugation. Spike and receptor interaction studies by S1-Fc binding analysis. HEK293T cells were 197 transfected with either pCDNA-ACE2 or pCDNA-DPP4 plasmids. 24h post-transfection, spike 198 S1-Fc proteins (5µg/ml) of SARS, SARS-2 and MERS CoV and the cells were independently 199 treated with Spirulina and green tea extract for 90 min at 37°C. In the first set of experiments, 200 untreated spike S1-Fc proteins were added to pretreated cells followed by binding assay at 4°C. In 201 the next set of experiments, the preincubated extract -spike S1-Fc protein mixture was added to 202 the untreated cells and were processed for binding assay. In both the experiments, following 1h of 203 spike S1-Fc incubation at 4°C, the cells were stained with Goat anti-human IgG conjugated with 204 FITC (1:300 dilution; A80-119F, Bethyl) subsequently counterstained with DAPI (Sigma Aldrich 205

Cat no D9542-10MG). Confocal images were acquired using Zeiss LSM 880 confocal laser-206 scanning microscope with an objective at 63x oil. Imaging parameters were kept the same for 207 control versus treated samples. The images were processed using the Zeiss ZEN blue software. 208 neutralizes SARSpv and also cross neutralizes SARS-2pv but not MERSpv (Fig 2a) whereas, 236 MERS-CoV polyclonal specifically neutralizes MERSpv but not SARSpv or SARS-2pv (Fig. 2b) , 237

suggesting that antibodies specifically bind on the receptor binding S1 domain of the CoVpvs and 238 neutralize the virus infection. It is well known that SARS and SARS-CoV-2 use human ACE2 as 239 the entry receptor to initiate an infection cycle [33] - [35] . To confirm that the CoVpv entry is 240 mediated by its specific receptor, we produced a recombinant soluble form of Angiotensin-241 converting enzyme 2 (sACE2) in HEK293T cells and purified using Ni-NTA affinity column. A 242 90 kDa band was observed in SDS PAGE and was confirmed by western blotting using ACE2 243 specific antibody (Fig. 2c) . Incubation of sACE2 with CoVpvs blocked the entry of SARSpv and 244 SARS-2pv but did not affect MERSpv infection (Fig 2d) . 245

Further, to confirm the receptor-mediated entry of the pseudotyped coronaviruses, we cloned the 246 complete ACE2 or MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) and was transiently 247 expressed on the non-susceptible BHK21 cells. The surface expression of ACE2 and DPP4 was confirmed by immunostaining using ACE2 and DPP4 polyclonal antibodies (Fig 2e) . Next, we 249 expressed the S1 domain of different spike proteins fused to the Fc domain of human IgG (S1-250 hFc) in HEK293T cells, yielding recombinant proteins of approximately 140 kDa, which was 251 confirmed by western blotting (Fig 2c) and immunostaining ( Supplementary Fig 1) . Incubation of 252 recombinant SARS-, SARS-2 -S1-hFc fusion proteins enables binding on hACE2 whereas MERS-253 S1-hFc bind on the hDPP4 transfected cells and no binding was observed in empty plasmid 254 transfected cells (Fig 2e) . Subsequently, ACE2 transiently expressed on the surface of non-255 susceptible BHK21 cells enable SARS-and SARS-2 pvs infection, whereas MERSpv or VSVpv 256 did not (Fig. 2f) . Similarly, MERS CoVpv infection was observed in hDPP4 expressed BHK21 257 cells, whereas SARS-, SARS-2-, and VSV-pvs infections were similar to empty plasmid 258 transfected cells (Fig 2g) . These data confirm that the developed pseudotyped CoVs use its specific 259 receptor to enter into the host cells. Interestingly, a significant reduction in infection was observed in all pvs pretreated with the 272 extracts (Fig 3b, d) but not in any of the preincubated cells (Fig 3a, c) . The results suggest that 273 CWE of Spirulina and Green tea inhibits pseudotyped coronavirus entry by attachment to 274 pseudovirus envelope protein rather than to the cell surface. 275

Next, to confirm that the observed inhibitory activity of the extracts was not due to proteolytic 276 cleavage of the spike glycoprotein, we incubated the spike S1-hFc protein with the extracts for 90 277 minutes and then the proteins were analyzed by western blotting. No protein degradation was 278 observed in any of the proteins in comparison to input (Supplementary 2). Next, we performed a 279 concentration-dependent inhibition assay for all pvs. We incubated pvs with varying 280 concentrations ranging from 0.1mg/ml -0.8mg/ml of Spirulina and green tea extracts. All three 281

CoVpvs were confirmed to be inhibited by CWE of Spirulina and Green tea with higher efficiency. 282 90% inhibition was observed in Spirulina treated SARSpv , SARS-2pv ,MERSpv and VSVpv 283 infection at 0.6mg/ml,0.5mg/ml,0.8mg/ml and 0.6 mg/ml respectively (Fig.4a) whereas green tea 284 treated pseudotyped viruses were inhibited at 0.1mgml, 0.12mg/ml, 0.1mg/ml and 0.25mg/ml 285 respectively (Fig.4b) . Most importantly, Green tea CWE showed more inhibitory activity on 286 pseudotyped coronaviruses in comparison to Spirulina extract. 287

To understand whether the inhibitory mechanism of Spirulina and green tea extracts are mediated 288 through inhibition of the interaction of spike and its receptor, we performed an S1 binding assay 289 on HEK293T cells expressing either ACE2 or DPP4. Cells were preincubated with the extracts 290 followed by the addition of S1 spike proteins at 4ºC. Binding assay of spike (S1-hFc) proteins was 291 analyzed by immunostaining followed by confocal microscopy. Similarly, we incubated the spike 292 S1 proteins with the extracts and then the mixture was added on the ACE2 or DPP4 cells. Cells 293 treated with either of the extracts did not inhibit the interaction of S1 and its receptor ACE2 or 294 DPP4 (Fig.5a) . In contrast, all three S1 proteins incubated with the extracts of green tea is unable 295 to bind to its receptor whereas Spirulina treated S1 proteins did not inhibit S1 -ACE2 or DPP4 296 interaction (Fig.5b) suggesting that the Spirulina extract blocks the viral entry through an unknown 297 mechanism. However, green tea extract binds to the S1 domain of the spike and prevents the spike 298 receptor interaction. Further studies with live viruses are required to elucidate the exact 299 mechanism of antiviral action. 300 301

The recent emergence of coronaviruses has caused serious global threats initiating diverse 303 approaches for biocontainment of these pathogens. Most of these emerging viruses are highly 304 pathogenic with limited therapeutic strategies and require BSL3/BSL4 facilities to handle these 305 pathogens. The ongoing pandemic of SARS CoV-2 which has spread across the world, is a notable 306 example that elucidates the need of diagnostics and robotic screening systems for identification of 307 antiviral compounds. However, the limited high-level biosafety equipped laboratories particularly 308 in developing countries inculcate the need for adapting to alternative approaches which can lead 309 to the handling of the viruses in a BSL2 facility. Here we report the production of VSV 310 pseudotyped virus particles for SARS CoV-2, SARS CoV, and MERS CoV. The developed 311 pseudoviruses were susceptible for infection in Vero E6 cells and other cells. We obtained a high 312 titer for SARS and MERS CoVpv whereas SARS CoV-2pv had a less titer which could be due to 313 less packaging efficiency in HEK293T cells which was consistent with the previous report [31] . 314

Pseudovirus production in Vero E6 cells [31] and expression of TMPRSS2 might enhance 315 pseudovirus production and infection [33] . The developed pseudotyped viruses could be efficiently 316 neutralized by specific polyclonal antibodies. SARS specific polyclonal neutralizes SARSpv and SARS-2pv as shown elsewhere [35] whereas MERS polyclonal antibodies neutralized MERSpv 318 [36] . Therefore, pvs are useful tools for studying virus neutralization assay for highly pathogenic 319 viruses in BSL2 facility [9] . Next, we showed that pseudoviruses specifically bind its receptor and 320 enter into host cells. Previous studies report that pvs can be used to understand the entry of 321 pathogenic viruses including coronaviruses [33] . previous studies, VSV entry is inhibited whereas cold water extracted Spirulina could inhibit the 329 entry of VSV. However, hot water extract of Spirulina could not inhibit VSV entry [38] which 330 might be due to the degradation of the major active compounds of Spirulina. but not hot water 331

extract. Next, we tested the antiviral activity of Spirulina and Green tea cold water extract on 332 pseudotyped coronaviruses. Treatment of cells with the extract did not inhibit viral entry whereas 333 preincubation of pseudoviruses with the extracts inhibited viral entry on Vero E6 cells suggesting 334 that the active compounds from Spirulina and green tea binds on the virus glycoprotein and block 335 the virus entry. To further understand the exact inhibitory mechanism, we performed an S1 binding 336 assay on DPP4 or ACE2 expressing cells using a mixture of S1 proteins preincubated with the 337 Spirulina and green tea extracts. Interestingly, all three S1 proteins treated with green tea abrogate 338 the interaction of spike with its receptor whereas Spirulina treated S1 proteins could bind to its 339 cellular receptor. The observed results suggest that green tea binds to the spike S1 domain and inhibits the viral entry. However, we speculate that EGCG, EGC, ECG, or EC, the most active 341 components in Green tea might be inhibiting the coronaviral entry not only binding exclusively to 342 S1 but also other domains of the viral envelope glycoprotein. These compounds are 

Emerging and Neglected Infectious Diseases: Insights, Advances, and 374 Challenges

Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses

The SARS epidemic in Hong Kong

Evidence for Camel-to-Human Transmission of MERS Coronavirus

A Novel Coronavirus from Patients with Pneumonia in China

Development of therapeutics for treatment of 387 Ebola virus infection

Antiviral potentials of medicinal plants

Antiherpetic activities of flavonoids against 393 herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)in vitro

Anti-HSV-1 activity of sulpoquinovosyl diacylglycerol isolated 396 from Spirulina platensis

Additional Inhibitory Effect of Tea Extract on the Growth of Influenza 399 A and B Viruses in MDCK Cells

Sulfated polysaccharides are potent 402 and selective inhibitors of various enveloped viruses, including herpes simplex virus, 403 cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus

Identification of natural compounds with antiviral activities against SARS-407 associated coronavirus

Antiviral effect of diammonium glycyrrhizinate and lithium 413 chloride on cell infection by pseudorabies herpesvirus

Molecular docking based screening of 416 neem-derived compounds with the NS1 protein of Influenza virus

Evaluation of antiviral activity of Ocimum sanctum and Acacia 419 arabica leaves extracts against H9N2 virus using embryonated chicken egg model

A review of the antiviral role of green tea catechins

Functional properties and health benefits of bioactive peptides derived 426 from Spirulina : A review

Calcium spirulan, an inhibitor of 429 enveloped virus replication, from a blue-green alga Spirulina platensis

Green Tea: Nature's Defense against Malignancies

Inhibitory effects of 435 (−)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 436 (HIV-1)

Epigallocatechin Gallate Inhibits the HIV Reverse 439 Transcription Step

Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C 442 virus

The green tea polyphenol, epigallocatechin-3-gallate, inhibits hepatitis C 445 virus entry

Antiviral effect of catechins in green tea on 448 influenza virus

Epigallocatechin gallate, the main component of tea polyphenol, binds to 451 CD4 and interferes with gp120 binding

The green tea 454 catechin, epigallocatechin gallate inhibits chikungunya virus infection

Dipeptidyl peptidase 4 is a functional receptor for the emerging human 457 coronavirus-EMC

Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular 460 stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

Generation of VSV pseudotypes using recombinant ΔG-VSV for studies on 463 virus entry, identification of entry inhibitors, and immune responses to vaccines

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is 466 Blocked by a Clinically Proven Protease Inhibitor

Angiotensin-converting enzyme 2 is a functional receptor for the SARS 469 coronavirus

A pneumonia outbreak associated with a new coronavirus of probable bat 472 origin

Novel antiviral agents: A medicinal plant perspective

A natural sulfated polysaccharide, calcium 477 spirulan, isolated from Spirulina platensis: In vitro and ex vivo evaluation of anti-herpes 478 simplex virus and anti-human immunodeficiency virus activities

Antiviral activity of Spirulina maxima against herpes simplex virus type 2

The olive leaf 485 extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus 486 (VHSV)

Effects of the olive-derived polyphenol oleuropein on human health

Effect of glycyrrhizin, 491 an active component of licorice roots, on HIV replication in cultures of peripheral blood 492 mononuclear cells from HIV-seropositive patients

Water extract of licorice had anti-viral activity against human respiratory 496 syncytial virus in human respiratory tract cell lines

Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated 500 coronavirus

Virucidal 503 activity of polysaccharide extracts from four algal species against herpes simplex virus

Well-tolerated Spirulina extract inhibits influenza virus replication and 507 reduces virus-induced mortality

Theaflavin derivatives in black tea and catechin derivatives in green tea 510 inhibit HIV-1 entry by targeting gp41

Epigallocatechin gallate inactivates clinical isolates of herpes simplex 513 virus

A Small Molecule Inhibits Virion Attachment to 516

Heparan Sulfate-or Sialic Acid-Containing Glycans

Inhibition of enterovirus 519 71-induced apoptosis by allophycocyanin isolated from a blue-green algaspirulina 520 platensis

Calcium spirulan derived from Spirulina platensis inhibits herpes simplex 522 virus 1 attachment to human keratinocytes and protects against herpes labialis

Antiviral activity of Arthrospira-derived spirulan-like substances

Induction of sulfated 527 polysaccharides in Spirulina platensis as response to nitrogen concentration and its 528 biological evaluation

Inhibition of 531 adenovirus infection and adenain by green tea catechins

Differential inhibitory effects of some catechin derivatives on the 534 activities of human immunodeficiency virus reverse transcriptase and cellular 535 deoxyribonucleic and ribonucleic acid polymerases

The evaluation of catechins that contain a galloyl moiety as potential HIV-538 1 integrase inhibitors

The green 541 tea molecule EGCG inhibits Zika virus entry

Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus 544 entry

Inhibition of Epstein-Barr virus lytic cycle by (-)-epigallocatechin 546 gallate

h post-transfection cells were independently incubated with PBS for 30 minutes followed by 663 spike S1-fc protein (both left panels), spirulina and green tea extracts (0.4mg/ml) followed by 664 S1-Fc protein (middle panels) and preincubated mixture of S1-Fc and Spirulina or green tea 665 extract (right panels). Binding of S1-Fc was immunostained by Goat anti-human IgG conjugated 666 with FITC and the binding of S1-Fc proteins were visualised by confocal microscopy. Green-667