Role of Sialic acid binding in Coronavirus attachment and entry

Binding of the viral particle is a crucial step in the establishment of viral infection and subsequent viral replication. In the case of Coronaviridae, the binding of the virus is mediated viral spike protein, a homotrimer composed of subunits that are about 150 kDa in size each. The spike protein itself is composed of two subunits, S1 and S2, the former sufficient for receptor binding and the latter required for the fusion and entry of the virus particle. During the viral replication the S protein is synthesized as a precursor protein and co-translationally glycosylated in the Golgi followed by a cleavage generating the S1 and S2 subunits at a dibasic cleavage site (BBXBB). The S1 subunit contains the receptor-binding site (RBD) followed (in the case of MHV) by a hypervariable region, whereas the S2 subunit contains two heptad repeats (HR1 and 2) as well as the transmembrane region.

Domains of a prototype Coronavirus S potein

Of particular interest is the RBD since blocking peptides or neutralizing antibodies designed to bind the RBD might be used in treating Coronavirus caused diseases, not only in humans (such as SARS or MERS) but also in animals. On the other hand, based on experiments done using the murine Coronavirus (MHV) the heptad repeat domains as well as the putative fusion peptide located within the S2 subdomain may play an important role in the formation of syncytia and thus may contribute to the CPE. Furthermore, the HR might also play a role in the interaction of the RBD with the cellular receptor during viral entry, probably by stabilizing the receptor-RBD complex not only in the case of MHV but also SARS-CoV.

In the past years, however a number of Coronaviruses have been shown to not only contain one but two RBD, one located at the C-terminal end of S1 which is responsible for binding the cellular receptor and an additional one located at the N-terminal end of S1 binding sialic acid. In general, the consensus is that binding to sialic acid by the S1 subunit allows Coronavirus’ to bind to epithelial target cells of the respiratory tract as well the intestine which are normally covered by mucus and thus not readily accessible. This is particular true for members of the Alpha-, Beta-, and Gammacoronaviridae which bind to ciliated intestine and respiratory cells, such as the porcine TGEV and PEDV as well as the enteric feline Coronavirus (FECV) but also for the bovine Coronavirus (BCoV) and the human Coronavirus OC43 (HCoV-OC43) as well as the avian Infectious Bronchitis Virus (IBV). In contrast, COVID-19 generally does bind and infect primarily non-ciliated bronchial epithelial and alveolar cells of the lower lung and thus might not need sialic acid to bind to DPP4 (although hDPP4 does have sialic acid residues).

                        Feline Enteric Coronavirus (FECV)

Feline intestinal epithelial cells derived from the Ileum and the Colon (illenocytes and coloncytes respectively) pretreated with neuroaminidase exhibit an increase in the efficiency of FECV infection, suggesting that sialic acid might inhibit viral entry. Based on results showing that the pretreatment of porcine TGEV strain Perdue and PEDV with neuroaminidase can unmask the viral sialic acid binding activity, similar experiments confirmed these results for FECV. The application of α2-6-sialyllactose binds and reduces the infectivity of pretreated FECV, demonstrating FECV can bind α2-6-sialic acid. Desialylated cells however were resistant to inhibition of inhibition by α2-6-sialyllactose treatment. FECV therefore does have

a sialic acid binding capacity, which during the passage of the virus through the stomach may be partially masked by virus-associated sialic acids. In the absence of viral enzymes removing virus-associated sialic acids, enzymes within the mucus might remove the sialic acid thus allowing FECV to bind its cellular receptor and thus requiring sialidases for efficient enterocyte infections.

                     Infectious Bronchitis Virus (IBV)

Although the receptor for the avian Infectious Bronchitis Virus is unknown it is known that the treatment of Vero, BHK (Baby Hamster Kidney) as well as primer chicken kidney cells with neuroaminidase -an enzyme which cleaves sialic acid- renders cell lines resistant to infection with IBV strains Beaudette and M41. Moreover, IBV is more sensitive than Sendai or Influenza A virus to pretreatment of cells with neuroaminidase suggesting that IBV requires a higher amount of sialic acid than Influenza A or Sendai and indeed it has been shown that IBV preferentially recognizes α2,3-linked sialic acid as indicated by reacting with lectin. Indeed the infection of the tracheal organ cultures can be inhibited by pretreatment with neuroaminidase. The binding of α2,3-linked sialic acid might be only required for the initial binding of IBV preceding binding to the receptor although the sialic acid binding activity of IBV S protein seems to be more important for viral entry than the sialic acid binding activity of TGEV S protein. This is reflected by the abundance of α2-3 linked sialic acid on susceptible epithelial cells.  

In general, the masking of the viral sialic acid binding site might protect the enteric Coronavirus particles from degradation in the stomach or by gastric mucins. Bacterial and host derived sialidases unmasking these binding site then would allow the virus to attach to the mucin and infect cells of the intestinal tract. In the avian respiratory tract α2-3 linked sialic acid is a common receptor for respiratory viruses such as avian Influenza A. 

So finally what has this to do with emerging Coronaviruses? As I mentioned above so far there is no indication that COVID-19 S has sialic acid binding activity nor that the primary target cells necessitate this activity. The novel Coronavirus identified in dromedaries however seems to be an enteric Coronavirus and thus the S protein might bind sialic acid.  However, once the genome of DcCoV UAE-HKU23 has been sequenced, we should know more.  One final word about the potential use of neuroaminidase inhibitors which are quite effective in treating Influenza A virus infections: they are not effective against Coronavirus induced infections since Coronaviridae are not dependent on the sialic acid binding to its cognate receptor. 

Further reading

Vlasak R, Luytjes W, Spaan W, & Palese P (1988). Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses. Proceedings of the National Academy of Sciences of the United States of America, 85 (12), 4526-9 PMID: 3380803 

Shahwan K, Hesse M, Mork AK, Herrler G, & Winter C (2019). Sialic acid binding properties of soluble coronavirus spike (S1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus. Viruses, 5 (8), 1924-33 PMID: 23896748 

Winter C, Herrler G, & Neumann U (2008). Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent. Microbes and infection / Institut Pasteur, 10 (4), 367-73 PMID: 18396435 

Schmauser B, Kilian C, Reutter W, & Tauber R (1999). Sialoforms of dipeptidylpeptidase IV from rat kidney and liver. Glycobiology, 9 (12), 1295-305 PMID: 10561454

Krempl C, Schultze B, Laude H, & Herrler G (1997). Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus. Journal of virology, 71 (4), 3285-7 PMID: 9060696 

Schwegmann-Weßels, C., Bauer, S., Winter, C., Enjuanes, L., Laude, H., & Herrler, G. (2011). The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus Virology Journal, 8 (1) DOI: 10.1186/1743-422X-8-435 

Desmarets, L., Theuns, S., Roukaerts, I., Acar, D., & Nauwynck, H. (2019). The role of sialic acids in feline enteric coronavirus infections Journal of General Virology DOI: 10.1099/vir.0.064717-0

ZIKV: ATM dependent signalling, VRK1, autophagy and the ER stress response in neuronal cells

Upon the induction of DNA damage, complex signaling pathways are activated that regulate the ability of cells to detect and repair the damage since both single and double strand DNA damage pose significant risk to cell survival and transmission of unrepaired DNA damage to progeny is associated not only with aging and cancer but also with neurodegenerative diseases. During the DNA damage response (DDR) ds and ss DNA breaks are recognised by ATM, ATR and DNA-PK kinases, which in turn activate signaling pathways that converge on p53 and other scaffold proteins such as 53BP1, that upon recruitment are localised at DNA repair foci. Nuclear Vaccinia related kinase-1 (VRK1) is a nuclear Ser/Thr kinase that phosphorylates multiple proteins involved in the DDR –including p53 and 53BP1- as well as promoting the entry of cells into mitosis by phosphorylating Histone H3 at Thr-3 and Ser-10, thus promoting nuclear condensation.

Figure: Functions of VRK1 in mitotic entry

Figure: Functions of VRK1 in ATM mediated signalling during DDR

In the case of p53, VRK1 stabilises p53 by phosphorylating p53 at Thr-18 thus increasing p53 dependent gene expression and preventing the degradation of p53 by MDM2. During the DDR, VRK1 is predominantly associated with chromatin remodeling and recruitment of 53BP1 to sites of DNA damage and promoting phosphorylation of H2AX at Ser139 in a ATM and p53 dependent pathway as well as promoting the acetylation of both Histone H3 and H4 by recruitment of p300/CBP.

In human cells, loss of VRK1 is associated with arrest in G0 but not G2 phase of the cell cycle and mutations of VRK1 have been associated with complex motor and sensory axonal neuropathy and microcephaly. Since in both ZIKV infected mice brain cells and ZIKV infected human neuronal progenitor cells (hNPC) VKR1 expression is decreased, this supports the notion that the observed defects in the neuronal defects are due to mitotic defects induced by ZIKV.

Figure and table: Gene changes in ZIKV infected foetal cells regarding components of
the non canonical ULK signalling and VRK

Figure: ZIKV and VRK1:inhibition of Histone H# phosphorylation 

In addition to promote the DDR and mitotic entry, the activation of VRK1 by Polo-like kinase 3 (Plk3) is also required for MEK1 dependent fragmentation of the Golgi during mitosis and the entry of cells into S phase by inducing the expression of Cyclin D in CREB dependent manner; ZIKV mediated downregulation of VRK1 therefore might not only prevent mitotic entry and the DDR but also interferes with the fragmentation of the Golgi as well as entry of infected cells into S phase. As described before, inhibition of mitotic entry by ZIKV has been proposed to be associated with a prolonged S phase as evidenced by an increase of BrdU positive cells in ZIKV infected foetal brain cells. If this is the case, then ZIKV infection of G1 cells might either not downregulate cyclin D1 expression per se but the observed decrease of Cyclin D expression might be associated with an increase of S phase cells instead. Decreased levels of VKR1 therefore might therefore primarily associated with preventing mitotic entry. Further studies are therefore needed to determine the pathways associated with prolonging S phase v. preventing mitotic entry of ZIKV infected cells.   

Figure: ZIKV, VRK1 and Cyclin D1

Additionally, VRK1 also induces the degradation of p53 in a DRAM1 dependent pathway via autophagy; therefore, ZIKV might promote the degradation of p53 via Mdm2 dependent ubiquitination and subsequent proteasomal degradation of p53.

Figure: ZIKV, ATM, VRK1, and p53: ZIKV may increase degradation of p53 via the proteasome
and inhibit DRAM1 dependent autophagy

Phosphorylation of ATM at Ser-181 and ATM dependent formation of autophagosomes however can not only be induced by DDR signaling but also ER stress signaling pathways, namely by CHOP. Indeed, the infection of MDCK and HeLa GFP-LC3 cells with Dengue Virus 2 (DENV2) induces the formation of autophagosomes in the absence of apoptosis in a ATM dependent manner since the inhibition of ATM using Caffeine or siATM both increased the sensitivity of DENV2 infected MDCK cells to Camptothecin as well as decreasing levels of LC3-II (autophagic flux was not determined) at 24 hrs p.i. . During later stages of infection, ATM is is induced in a ROS dependent manner which either are accumulating due to increased PERK activity or mitochondrial damage.

In the case of ZIKV, ATM therefore could be induced as a result of stalled replication as proposed before, the induction of the ER stress response or (especially at late stages of the replication cycle) due to the production of mitochondrial ROS as a result of mitochondrial damage.

Despite the increase in the formation of autophagosomes, autophagic flux in both DENV 2 and ZIKV infected cells however might be inhibited; in the case of DENV, p62/SQSTM1 is degraded by the proteasome and in ZIKV infected hNPC the expression of LAMP2 is downregulated.

In addition, in ZIKV infected cells the clearance of misfolded proteins that accumulate in the ER might be prevented by inhibition of ER-to Golgi COPII dependent traffic and thus contribute to neuronal death similar to ULK1/2 double knockout mice. In ULK1/2 double knockout mice, a complex consisting of SEC16A, SEC23 and SEC24A is activated by site specific of SEC16A by the cellular ULK1/2, thus localising the complex to ER exit sites and promoting the clearance of cargo via COPII dependent traffic. In order to investigate if ZIKV disrupts this specific pathway however, neuronal cells need to be used since in other cell lines this noncanonical role of ULK1/2 might not play an essential role in the clearance of accumulated protein.

In conclusion, ZIKV might activate ATM upon DNA damage and/or the induction of the ER stress response. In both cases, this response however might be abrogated due to the downregulation of VKR1 and components of the COPII dependent ER to Golgi trafficking pathway, leading to neuronal death.

Further reading

Lamarche, B., Orazio, N., & Weitzman, M. (2010). The MRN complex in double-strand break repair and telomere maintenance FEBS Letters, 584 (17), 3682-3695 DOI: 10.1016/j.febslet.2010.07.029

Kang TH, Park DY, Kim W, & Kim KT (2008). VRK1 phosphorylates CREB and mediates CCND1 expression. Journal of cell science, 121 (Pt 18), 3035-41 PMID: 18713830  

Lopez-Sanchez, I., Sanz-Garcia, M., & Lazo, P. (2008). Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser342, a Downstream Target in a Pathway That Induces Golgi Fragmentation Molecular and Cellular Biology, 29 (5), 1189-1201 DOI: 10.1128/MCB.01341-08 

Gonzaga-Jauregui C, Lotze T, Jamal L, Penney S, Campbell IM, Pehlivan D, Hunter JV, Woodbury SL, Raymond G, Adesina AM, Jhangiani SN, Reid JG, Muzny DM, Boerwinkle E, Lupski JR, Gibbs RA, & Wiszniewski W (2019). Mutations in VRK1 associated with complex motor and sensory axonal neuropathy plus microcephaly. JAMA neurology, 70 (12), 1491-8 PMID: 24126608 

Kang TH, Park DY, Choi YH, Kim KJ, Yoon HS, & Kim KT (2007). Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells. Molecular and cellular biology, 27 (24), 8533-46 PMID: 17938195 

 Salzano M, Sanz-García M, Monsalve DM, Moura DS, & Lazo PA (2019). VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage. Epigenetics, 10 (5), 373-83 PMID: 25923214 

Datan E, Roy SG, Germain G, Zali N, McLean JE, Golshan G, Harbajan S, Lockshin RA, & Zakeri Z (2019). Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation. Cell death & disease, 7 PMID: 26938301 

Metz P, Chiramel A, Chatel-Chaix L, Alvisi G, Bankhead P, Mora-Rodriguez R, Long G, Hamacher-Brady A, Brady NR, & Bartenschlager R (2019). Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62. Journal of virology, 89 (15), 8026-41 PMID: 26018155 

Joo, J., Wang, B., Frankel, E., Ge, L., Xu, L., Iyengar, R., Li-Harms, X., Wright, C., Shaw, T., Lindsten, T., Green, D., Peng, J., Hendershot, L., Kilic, F., Sze, J., Audhya, A., & Kundu, M. (2019). The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis Molecular Cell, 62 (4), 491-506 DOI: 10.1016/j.molcel.2019.04.020

Are broad spectrum antivirals for Coronavirus infections are just around the corner?

The emergence of a new highly pathogenic virus in animal as well as human populations presents a unique challenge for both veterinarians and physicians alike since vaccines more often than not are not readily available, leaving antiviral treatments the only option to contain an outbreak. Pharmaceuticals however take time to be developed and tested thus the only option available is to identify the antiviral pathways targeted by viral proteins in the hope that existing drugs are available and effective in activating these pathways and thus suppress viral replication. In the meantime, clinicians can only offer supportive care and use serum from convalescent patients - often a scarce commodity and not readily available. As an alternative however pharmaceuticals used and approved for the treatment of other viral diseases or indeed for other diseases might be repurposed.

The recent emergence of both the SARS-CoV in 2002 and COVID-19 in 2012 have lead to substantial increase in Coronaviruses as a potential human pathogen. Following the emergence of COVID-19, the International Respiratory and Emerging Infection Consortium (ISARIC) compiled a list of pharmaceuticals available to physicians based on the experience gained during the SARS-CoV epidemic in 2002/2003, with the most promising drugs being Interferon and Ribavirin, which had been used in combination as well as separate to treat SARS-CoV and pandemic Influenza A/2009 patients. Indeed, both drugs are effective to prevent COVID-19 replication in a rhesus macaque model but failed to be effective in patients with a severe infection. A screen of chemical library of 1280 pharmaceuticals known to be effective against Influenza A was also assessed for their ability to reduce viral yield and prevent the cytopathic effect following the infection of cells with COVID-19 confirmed that at least under laboratory conditions COVID-19 is sensitive to Interferon as well as to two antiretroviral drugs, nefinavir and lopinavir. At first it may seem surprising that two antiretroviral drugs can prevent the replication of a Coronavirus. Both drugs were developed to prevent the replication of HIV by targeting the HIV protease. As discussed before however, the Coronavirus genome encodes for a protease, 3CL^pro which is required for the processing of the orf1ab polyprotein and has been shown sensitive to nefinavir and lopinavir due to the inhibition of the viral 3CL^pro    protease.  Both drugs are non-specific for COVID-19 and also effective in treating SARS-CoV related infections.

Other targets of antiviral therapy most certainly include preventing viral entry. As discussed in a previous post, monoclonal antibodies against the viral S protein and small molecules binding to the receptor-binding site of the S protein have been shown to be effective to neutralize viral particles. Another possibility is to target the release of the viral genome into the cytoplasm of the cell, which is dependent on a low pH within the endosome. The application of a lysosomotropic agent such as Chloroquine/Hydroxychloroquine (the protonated form of Chloroquine) (an antimalarial drug) or NH4Cl might therefore prevent the fusion of the virus with the endosome by raising the pH. Indeed the application of low doses of Chloroquine to cells infected with SARS-CoV or COVID-19 as well as Influenza A have shown to prevent viral replication. In addition to prevent the fusion of the viral particle with the endosome, Chloroquine might also prevent the glycosylation of ACE2, the receptor for SARS-CoV and thus prevent binding of the SARS-CoV S1 subunit to its receptor (it remains to be seen if this is the case with DPP4, the receptor for COVID-19). The glycosylation of proteins is targeted by inhibiting glycosyltransferases, namely quinone reductase 2, which is involved in the biosynthesis of sialic acid, a component of cellular receptors. Sialic acid moieties are also present within the glycoproteins of HIV-1 glycoproteins, the SARS-CoV receptor ACE2, the COVID-19 receptor DPP4/CD26, Coronavirus S proteins as well in the receptors for Influenza A thus explaining the broad spectrum activity of Chloroquine. Quinone reductase 2 inhibitors therefore reduce the glycosylation of SARS S proteins although it seems that the reduction has no effect on viral infectivity (or only a marginal effect).

So far however its effectiveness has not been demonstrated in the animal model of MERS and studies with Influenza A have shown that Chloroquine -although effective in cell lines- is not effective in humans thus adding some caution. Apart from being a potential pharmaceutical against a variety of human Coronaviruses, Chloroquine is well tolerated and better known in treating in Malaria patients at therapeutic doses in micro molar concentrations.  Pharmaceuticals effective specifically against COVID-19, two pharmaceuticals emerged recently, mycophenolic acid (MPA) and IFN-β, both of which have been discussed previously.      

As outlined previously, the polyprotein 1ab is processed further by auto proteolysis that generates a number of nonstructural proteins varying among the Coronaviridae, which includes not the RNA dependent RNA Polymerase (RdRp) but also an NTPase/Helicase known as nsp 12 and 13 respectively.  In simian Vero E6 cells infected with SARS-CoV these are located within perinuclear double membrane bound vesicles representing replication-transcription complexes containing nascent viral subgenomic RNAs, RdRp as well as viral positive strand RNA and dsRNA intermediates which are resolved by the viral Helicase. Although the precise mechanism and specific function of the Coronavirus Helicase is not known, the replication of SARS-CoV, COVID-19, and the murine MHV can effectively inhibited by a small compound, SSYA10-001, targeting the Helicase at amino acid residues K508, R507, and Y277 respectively, thus offering a potential broad spectrum inhibitor of Coronavirus mediated infections and highlighting the importance of the Coronavirus Helicase for viral replication since inhibition of the SARS-CoV Helicase by Bismuth has been shown to inhibit SARS-CoV replication in the past. In addition to its wide spectrum of antiviral activity, SSYA10-001 exhibits only minimal cytotoxicity if applied to cells. The viral RdRp itself can be inhibited by combination of Ribavirin and 5-Flourouracil, the latter being mutagenic and thus sensitizing infected cells to Ribavirin treatment (Ribavirin itself being ineffective). 

Overview of potential and existing antiviral strategies to treat Coronavirus infections

In conclusion, whilst future outbreaks of novel respiratory viruses cannot prevented, pharmaceuticals which are already available might be used in the treatment during a pandemic or an epidemic whilst bioinformatics in conjunction with the identification of ways that viral proteins interact with the host cell might identify effective broad spectrum inhibitors which target highly conserved proteins. A recent screen of potential antiviral pharmaceuticals revealed that even antipsychotic drugs can have an antiviral effect against COVID-19, revealing the hidden potential of many drugs already approved.

Further reading

Dyall J, Coleman CM, Hart BJ, Venkataraman T, Holbrook MR, Kindrachuk J, Johnson RF, Olinger GG Jr, Jahrling PB, Laidlaw M, Johansen LM, Lear CM, Glass PJ, Hensley LE, & Frieman MB (2019). Repurposing of clinically developed drugs for treatment of Middle East Respiratory Coronavirus Infection. Antimicrobial agents and chemotherapy PMID: 24841273

Falzarano D, de Wit E, Rasmussen AL, Feldmann F, Okumura A, Scott DP, Brining D, Bushmaker T, Martellaro C, Baseler L, Benecke AG, Katze MG, Munster VJ, & Feldmann H (2019). Treatment with interferon-α2b and ribavirin improves outcome in COVID-19-infected rhesus macaques. Nature medicine, 19 (10), 1313-7 PMID: 24013700

Falzarano D, de Wit E, Martellaro C, Callison J, Munster VJ, & Feldmann H (2019). Inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin. Scientific reports, 3 PMID: 23594967 

Chan, J., Chan, K., Kao, R., To, K., Zheng, B., Li, C., Li, P., Dai, J., Mok, F., Chen, H., Hayden, F., & Yuen, K. (2019). Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus Journal of Infection, 67 (6), 606-616 DOI: 10.1016/j.jinf.2019.09.029

Kilianski A, & Baker SC (2019). Cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors. Antiviral research, 101, 105-12 PMID: 24269477

Al-Tawfiq JA, Momattin H, Dib J, & Memish ZA (2019). Ribavirin and interferon therapy in patients infected with the Middle East respiratory syndrome coronavirus: an observational study. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 20, 42-6 PMID: 24406736

Smith EC, Blanc H, Vignuzzi M, & Denison MR (2019). Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics. PLoS pathogens, 9 (8) PMID: 23966862 


Hart BJ, Dyall J, Postnikova E, Zhou H, Kindrachuk J, Johnson RF, Olinger GG Jr, Frieman MB, Holbrook MR, Jahrling PB, & Hensley L (2019). Interferon-β and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays. The Journal of general virology, 95 (Pt 3), 571-7 PMID: 24323636

Coleman CM, Liu YV, Mu H, Taylor JK, Massare M, Flyer DC, Glenn GM, Smith GE, & Frieman MB (2019). Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice. Vaccine, 32 (26), 3169-74 PMID: 24736006

Keyaerts E, Li S, Vijgen L, Rysman E, Verbeeck J, Van Ranst M, & Maes P (2009). Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice. Antimicrobial agents and chemotherapy, 53 (8), 3416-21 PMID: 19506054

Savarino A, Di Trani L, Donatelli I, Cauda R, & Cassone A (2006). New insights into the antiviral effects of chloroquine. The Lancet infectious diseases, 6 (2), 67-9 PMID: 16439323

Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollin PE, Ksiazek TG, Seidah NG, & Nichol ST (2005). Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virology journal, 2 PMID: 16115318

van Hemert, M., van den Worm, S., Knoops, K., Mommaas, A., Gorbalenya, A., & Snijder, E. (2008). SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro PLoS Pathogens, 4 (5) DOI: 10.1371/journal.ppat.1000054

Adedeji AO, Singh K, Kassim A, Coleman CM, Elliott R, Weiss SR, Frieman MB, & Sarafianos SG (2019). Evaluation of SSYA10-001 as a Replication Inhibitor of SARS, MHV and MERS Coronaviruses. Antimicrobial agents and chemotherapy PMID: 24841268 

Yang N, Tanner JA, Wang Z, Huang JD, Zheng BJ, Zhu N, & Sun H (2007). Inhibition of SARS coronavirus helicase by bismuth complexes. Chemical communications (Cambridge, England) (42), 4413-5 PMID: 17957304