Artemisia Annua: A Potent Antimicrobial

Artemisia Annua: A Potent Antimicrobial

August 31, 2020

Artemisia Annua: A Potent Antimicrobial

by James Odell, OMD, ND, L.Ac.

Artemisia annua has been used in China for more than 2000 years to treat fevers and more recently used in the treatment of the chloroquine-resistant and cerebral malaria (Plasmodium falciparum). Much focus has now been paid to its effectiveness in the treatment of SARS-CoV-2 (Covid-19). Its ancient Chinese name Qing Hao literally means “green herb.” Qing Hao was mentioned in the ancient text (168BC) Wu Shi Er Bing Fang or “Recipes for Fifty-Two Ailments”, as a remedy for fevers. The genus Artemisia consists of over 400 species, many of which have an aromatic, bitter taste. Herbal extracts of Artemisia annua have been used for thousands of years in other parts of the world, particularly Southeast Asia, Africa, India, and South America, to treat malaria and a variety of infectious diseases. Apart from its anti-malarial properties, Artemisia annua has been used in traditional Chinese medicine to stimulate hair growth, to promote longevity, as a food additive, as an anti-inflammatory, as well as a treatment for numerous external illnesses including hemorrhoids, lice and boils. 


Botanical Aspects


Artemisia is a large, diverse plant genus with between 200 and 400 species and consists of hardy herbs and shrubs belonging to the Magnoliopsida class of flowering plants. Artemisia annua is an annual shrub of 50–150 cm in height. The shrub grows in temperate climates and is most widespread in China and Vietnam, but is also cultivated in East Africa, the United States, Russia, India, Brazil, and several other countries.1, 2 The reproduction of the shrub occurs by insects, self-pollination, and wind distribution.3


Artemisia annua Chemical Properties


The essential oil of Artemisia annua is rich in mono- and sesquiterpenes with numerous medicinal properties. Significant variations in its percentage and composition have been identified (main constituents may be camphor (up to 48%), germacrene D (up to 18.9%), artemisia ketone (up to 68%), and 1,8 cineole (up to 51.5%)). The oil has been subjected to numerous studies supporting exciting antiparasitic, antibacterial, antiviral, and antifungal activities. One of the more medicinal components found in Artemisia annua is artemisinin, first isolated in China in 1971.4


Artemisinin is the constituent with the greatest antimalarial activity. Up to 42% of the total artemisinin content is found in the upper leaves, where it accumulates in the glandular trichomes of the leaves. Artemisinin has been found in only two other species, Artemisia apiacea and Artemisia lance 5, and since that time its efficacy against malaria has been amply demonstrated.6, 7, 8, 9, 10, 11


The total amount of artemisinin found in different varieties of Artemisia annua varies slightly depending on extraction methods, different collection periods, different sample preparation, and different environmental influences.12 The artemisinin content in the plant exhibits the highest quantities usually just before flowering. Except for Artemisia annua, artemisinin is also present in Artemisia apiacea and Artemisia lancea, but only in minor quantities.13


Nowadays, many researchers are still investigating the effect of artemisinin and its analogues on the malarial parasite (Plasmodium) by modifying the structure of peroxides, ethers and ozonides in artemisinin. This improves the killing rate of plasmodium parasites for both in vitro and in vivo models as well as a faster clinical response for humans.14


Antimalarial Mechanism of Action of Artemisia annua


Malaria is one of the most severe public health problems worldwide. It is a leading cause of death and disease in many developing countries, where young children and pregnant women are the groups most affected. Worldwide an estimated 450,000 deaths annually (around 1200 per day) are attributed to malaria. This infection is caused primarily by the Plasmodium falciparum parasite, which largely reside in red blood cells and contains iron-rich heme-groups (in the form of hemozoin). Such hematophagous organisms digest hemoglobin and release high quantities of free heme, which is the non-protein component of hemoglobin. As a result, hemozoin pigment and other toxic factors such as glycosylphosphatidylinositol (GPI) are also released into the blood. These products, particularly the GPI, activate macrophages and endothelial cells to secrete cytokines and inflammatory mediators such as tumor necrosis factor, interferon-γ, interleukin-1, IL-6, IL-8, macrophage colony-stimulating factor, and lymphotoxin, as well as superoxide and nitric oxide. These inflammatory cytokines and mediators can cause significant damage to organs and tissues.15, 16, 17


The parasite is fairly shielded from attack by the body’s immune system since it resides within the liver and blood cells for much of its human life cycle and is relatively invisible to immune surveillance. However, circulating infected blood cells are destroyed in the spleen. To avoid this fate, the Plasmodium falciparum parasite displays adhesive proteins on the surface of the infected blood cells, causing the blood cells to stick to the walls of small blood vessels, thus sequestering the parasite from passage through the general circulation and the spleen. Sequestered red blood cells can breach the blood-brain barrier and cause cerebral malaria. Artemisinin is also active against other parasite species such as Toxoplasma and Babesia that do not contain hematin.


Chemically, artemisinin is a sesquiterpene lactone that contains an unusual peroxide bridge. This 1, 2, 4-trioxane ring of endoperoxide is responsible for the drug’s mechanism of action.  Thus, the proposed antimalarial mechanism of action of artemisinin involves cleavage of endoperoxide bridges by iron, producing free radicals (hypervalent iron-oxo species, epoxides, aldehydes, and dicarbonyl compounds) which damage biological macromolecules causing oxidative stress in the cells of the parasite.18, 19


Artemisinins have also been investigated for their anti-proliferative activity against a wide range of cancer cell lines. Artemisinin results in decreased proliferation, increased levels of oxidative stress, induction of apoptosis and inhibition of angiogenesis in cancer cells. Artemisinins have also been shown to inhibit the falcipains, a papain family cysteine protease that aids hemoglobin degradation.20




Over the last ten years as the worldwide demand for artemisinin has become evident, Chinese, Vietnamese, African, and Indian plant breeding institutes have developed high-yielding Artemisia hybrids. Factories were developed in all three countries to extract artemisinin and manufacture its anti-malarial compounds. East African factories are currently exporting artemisinin to pharmaceutical factories in India and Europe, where the final products are made.


Drug resistance is a growing issue for the treatment of malaria in the 21st-century. Resistance is now common against all classes of antimalarial drugs except for artemisinins. Artemisinin treatment of resistant drug strains has therefore become increasingly popular. Unfortunately, while the cost of cultivation and production of artemisia is lower than that of other competitive pharmaceuticals, politics involving the pharmaceutical industry have restricted their use in the developing world.

From Quinine to Chloroquine to Hydroxychloroquine to Artemisinin


In 1820, the first antimalarial drug quinine was extracted from cinchona bark by French pharmacists Pelletier and Caventou.21 In the 1940s, limited by the raw materials for quinine extraction, German scientists synthesized chloroquine, which is similar to natural quinine in chemical structure.

In 1950, chemists Alexander Surrey and Henry Hammer published the synthesis of hydroxychloroquine which was even more effective with less toxicity.

By the mid-20th century, malaria was gradually controlled in China. However, parts of Africa still suffer high epidemic proportions of malaria. In the 1960’s an epidemic broke out which spread rapidly in Southeast Asia and South America. In addition, the plasmodium falciparum parasite was developing a resistance to chloroquine and hydroxychloroquine. Inspired by ancient books of traditional Chinese medicine, Youyou Tu, a Chinese scientist, successfully extracted artemisinin from Artemisia annua. With a 100% inhibition rate against plasmodium, artemisinin is now used for chloroquine and hydroxychloroquine resistant malaria. For her work, Tu was awarded the 2011 Lasker Award in clinical medicine and the 2015 Nobel Prize in Physiology or Medicine jointly with William Campbell and Satoshi Ōmura. Tu is the first Chinese Nobel laureate in physiology or medicine and the first female citizen of the People’s Republic of China to receive a Nobel Prize in any category.22

Antiviral Effects of Chloroquine and Hydroxychloroquine


To better understand the therapeutic antiviral similarities of chloroquine derivatives and Artemisia annua extracts it is beneficial to review the antiviral background of chloroquine. Chloroquine has been confirmed to be effective during epidemics of various infectious diseases, especially Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome Coronavirus (MERS). In 2003, SARS broke out in China. According to the World Health Organization, a total of 8,098 people worldwide became sick with SARS during this outbreak, of whom 774 died. In 2004, MarcVan Ranst and colleagues found that chloroquine effectively inhibited SARS coronavirus replication in vitro.23


In 2005, Stuart Nichol and colleagues found that chloroquine suppressed SARS virus replication both before and after infection, suggesting a preventative and therapeutic role of chloroquine against SARS.24


In 2014 Eric Snijder and colleagues successfully inhibited MERS coronavirus replication by chloroquine in monkeys, with similar suppressive effect against SARS and human coronavirus.25 Additionally, chloroquine has also been reported to inhibit Human Immunodeficiency Virus (HIV), Zika virus (ZIKV), and dengue virus (DENV).26 Chloroquine phosphate was also reported to alleviate lung autophagy induced by avian influenza A (H5N1) and reduce alveolar injury in mice.27


February 2020, Wuhan Virus Research Institute of the Chinese Academy of Sciences and other units jointly published the results on cell research, which showed that chloroquine phosphate effectively inhibited SARS-Cov-2 and such inhibition was superior as compared to Remdesivir.28 Other recent studies have validated those findings.29, 30


The antiviral mechanisms of chloroquine and hydroxychloroquine on SARS-Cov-2 is proposed as follows:


1) To weaken the binding of the virus to the receptor by interfering with the terminal glycosylation of the receptor protein angiotensin 2 receptor invertase of coronavirus.

2) As an alkaline drug, chloroquine increases pH value inside endosomes which was not conducive for virus-cell fusion.

3) Inhibits cell autophagy and regulates host immune reaction to suppress viral infection and replication.

4) Suppresses transcription and translation of virus protein by binding to viral protease; and

5) Alleviates cytokine storm through inhibiting the production and release of TNF-α and IL-6.


In June 2020, the U.S. Food and Drug Administration revoked the emergency use authorization that allowed for chloroquine phosphate and hydroxychloroquine sulfate to be used to treat certain hospitalized patients with COVID-19 when a clinical trial was unavailable, or participation in a clinical trial was not feasible. More than 35 states have now restricted prescriptions for hydroxychloroquine, and at least five of those have rules specifically prohibiting prescribing the drug as a preventive measure. Fortunately, we still have artemisia extracts and other immunological nutrients (vitamin D3, zinc, vitamin C) available – for now.


Artemisia annua Extracts Effective Against Viruses


Due to its similar history to chloroquine in the treatment of malaria and viruses, scientists at several institutions have researched whether extracts of Artemisia annua – pure artemisinin and related derivatives – may be effective against the COVID-19 virus. These compounds would be attractive candidates for immediate use as they have an excellent safety profile, are readily available, and are relatively inexpensive.


Numerous in vitro studies have reported that artemisinins have antiviral properties. Artemisinins reduce replication rates of hepatitis B and C viruses 31, 32, a range of human herpes viruses 33, 34, 35, HIV-1 36, influenza virus A 37, 38, and a bovine viral diarrhea virus39, in the low micromolar range. 


Like chloroquine, Artemisia annua extracts have even shown significant activity against the SARS coronavirus. In 2003, Li and colleagues showed that artemisinin was effective in treating SARS-CoV in vitro.40 Since the beginning of the COVID-19 pandemic, formulations of Artemisia annua have been used in Africa, Madagascar, and China for both COVID-19 prevention and treatment.41


Last June 2020, chemists at the Max Planck Institute of Colloids and Interfaces (Potsdam, Germany) in close collaboration with virologists at Freie Universität Berlin demonstrated in laboratory studies that aqueous and ethanolic extracts Artemisia annua are active against the SARS-CoV-2 (COIVID-19) virus. Human clinical trials to test the efficacy of both teas and coffee containing Artemisia annua are about to begin at the University of Kentucky’s academic medical center.42 Artemisia annua leaves, from a cultivated seed line grown by ArtemiLife Kentucky, USA, when extracted with absolute ethanol or distilled water produces the strongest antiviral activity. The addition of either ethanolic or aqueous Artemisia annua extracts prior to the introduction of the virus resulted in significantly reduced plaque formation. The most active extract of both Artemisia annua and coffee was found to be ethanolic. However, artemisinin alone does not present much antiviral activity. “I was surprised to find that A. annua extracts worked significantly better than pure artemisinin derivatives and that the addition of coffee further enhanced the activity” says Klaus Osterrieder, Professor of Virology at Freie Universität Berlin who conducted all activity assays.


In SARS-CoV-2 (COVID-19), cellular adaptive immunity is primarily involved, in particular, CD8 and CD4 lymphocytes that stimulate the B lymphocytes responsible for the production of antibodies targeting the coronavirus. In addition, there is a cytokine storm in patients infected with SARS-CoV-2 which is responsible for a major inflammatory response and their very severe progressive clinical state. The increase in interleukin-10 and TNF alpha reduces CD4 counts, causes functional exhaustion of immune cells, and induces, at their site of action (liver, vascular endothelium), runaway production and action of inflammatory proteins, resulting in secondary aggravation of COVID-19 patients.


Artemisia annua has extensively recognized antiviral activity (anti HSV1, Poliovirus, RSV, hepatitis C anti-virus, type 2 dengue virus, hantavirus, human cytomegalovirus) and anti-HIV in vitro, due in partto the artemisinin, flavonoids, quercetin and dicaffeoylquinic acids it contains. These molecules have been shown to inhibit the enzymatic activity of CLPro (Chymotrypsin-like protease), an enzyme produced by SARS-CoV-2.


The antiviral action of Artemisia annua, which is achieved by stimulating adaptive immunity, regulating the production of the pro-inflammatory cytokines prostaglandin E2 (PGE2), IL-6, IL-10, TNF alpha, and increasing the genesis of CD4, CD8 and interferon gamma, involves many minerals and biomolecules: the properties of flavonoids, polyphenols, triterpenes, sterols, saponins, polysaccharides, artemisinin and its derivatives, the concentration of zinc, gallium and selenium in the plant play a role in the immune, antiviral, antioxidant and anti-inflammatory response.


The plant is rich in vitamins A and E, of which one, when supplemented, is known to reduce morbidity and mortality in viral infections, notably HIV among others, and the other is a powerful antioxidant. Therefore the combination of these biomolecules and the intake of Artemisia annua may strengthen the exhausted adaptive immunity and modulate the runaway inflammatory response during COVID-19 infection, as has already been demonstrated in other serious viral and parasitic infections.


As more research develops it is likely that Artemisia annua extracts will take their place as a first-line defense against coronaviruses. Given that this plant has been extensively used for more than 2000 years in traditional Chinese medicine for treatment of fever, viruses, and malaria, the evidence argues for the inclusion of inexpensive Artemisia annua dried leaf tablets, capsules, or teas into the arsenal of remedies to combat coronavirus.


Lastly, malaria treatment is more complicated for human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients. Malaria and HIV co-infection represents a major health burden in Africa, primarily because it is now well known that HIV infection results in a higher incidence and more severe manifestations of malaria. With a compromised immune system, AIDS patients are more susceptible to malaria and respond slower to malaria therapy. In several studies and in clinical observation, Artemisia annua has demonstrated anti-HIV activity.43, 44 Hence, the use of Artemisia annua not only treats malaria but should also enhance the well-being of HIV/AIDS patients.


Preparation and Dosage


Artemisia annua is typically prepared as a tea extracted with water according to the rules of traditional Chinese medicine. There have been few well-controlled studies investigating the extraction, recovery, and stabilization of artemisinin and other compounds in Artemisia annua tea infusions. A systematic study of preparations of Artemisia annua therapeutic tea infusion was performed by van der Kooy and colleagues.45 This study showed that nearly 93% of available artemisinin was extracted from dried Artemisia annua leaves, but only under certain conditions. The best preparation method was: 9 g dried leaves/L, for 5 min at 100 °C. Subsequent storage of the tea infusion at room temperature showed that the concentration of artemisinin was stable for more than 24 hours. This is important for malaria-endemic locations where there is no refrigeration. Other studies using the same extraction protocol also measured the extraction and stability of artemisinin and certain key flavonoids in the tea. Artemisinin was found to be stable at room temperature for up to 48 hours.46 However, some flavonoids were poorly extracted and not stable at room temperature, therefore it may be best to refrigerate after the infusion is complete.47


Clearly, if a tea infusion is to be a therapeutic option, it must be prepared and consumed consistently and reliably.  Artemisia annua is also available commercially in extracts such as capsules and tinctures. As with all herbal remedies, the correct dosage depends on a variety factors such as the illness treated, the age of the person, health status, and number of other conditions.


Herb-Drug Reactions


Artemisinin has no reported toxicity if taken in recommended doses for a limited period of time.51 In animal studies, artemisinin has been used in high oral doses in dogs and rabbits52 and at 200-300 mg/kg BW in mice53 without toxicity. Artemisinin has been effective against Plasmodium in humans at doses of approximately 30 mg/kg BW, but it has poor bioavailability and a short half-life that is quickly eliminated from the body.54, 55 Artemisinin derivatives (dihydroartemisinin, artesunate, artemether, arteether) present better bioavailability and antimalarial activity than artemisinin, but have different safety margins than artemisinin. The bioavailability and half-lives also vary with the delivery mechanism (intramuscular, intravenous, intraperitoneal, oral).




Evidence is mounting for the therapeutic effectiveness of the use of Artemisia annua not only in the treatment of malaria, but also for various viruses, including coronaviruses. The complex mixture of antiparasitic compounds in the plant appears to account for its therapeutic activity with the animal and human trials supporting this claim. It is also clear that the cost of using Artemisia annua is a fraction of that for any other existing or potential antimalarial or antiviral treatment. Considering that for more than 2000 years this plant was used in traditional Chinese medicine for the treatment of fever with little to no significant toxicity and no clear signs of artemisinin drug resistance. Thus, the cumulative evidence argues for the inclusion of Artemisia annua extracts,tinctures, and teas into the arsenal of remedies to not only combat malaria, but also numerous other diseases, particularly viruses.



1. Bhakuni, D.S., Goel, A.K., Jain, S., Mehrotra, B.N., Patnaik, G.K., Prakash, V., 1988. Screening of Indian plants for biological activity: part XIII. Indian Journal of Experimental Biology 26, 883–904.


2. Bhakuni, D.S., Goel, A.K., Jain, S., Mehrotra, B.N., Srimal, R.C., 1990. Screening of Indian plants for biological activity: part XIV. Indian Journal of Experimental Biology 28, 619–637.


3. Bown, Deni. The Royal Horticultural Society encyclopedia of herbs & their uses. Dorling Kindersley Limited, 1995.


4. Klayman, Daniel L. “Qinghaosu (artemisinin): an antimalarial drug from China.” Science 228, no. 4703 (1985): 1049-1055.


5. Tan, Ren Xhiang, W. F. Zheng, and H. Q. Tang. “Biologically active substances from the genus Artemisia.” Planta medica 64, no. 04 (1998): 295-302.


6. Hien, Tran Tinh, and Nicholas J. White. “Qinghaosu.” Lancet (British edition) 8845 (1993): 603-608.


7. Mueller, Markus S., I. B. Karhagomba, Hans Martin Hirt, and Emmanuel Wemakor. “The potential of Artemisia annua L. as a locally produced remedy for malaria in the tropics: agricultural, chemical and clinical aspects.” Journal of ethnopharmacology 73, no. 3 (2000): 487-493.


8. Mueller, Markus S., Nyabuhanga Runyambo, Irmela Wagner, Steffen Borrmann, Klaus Dietz, and Lutz Heide. “Randomized controlled trial of a traditional preparation of Artemisia annua L.(Annual Wormwood) in the treatment of malaria.” Transactions of the Royal Society of Tropical Medicine and Hygiene 98, no. 5 (2004): 318-321.


9. Balint, Gabor A. “Artemisinin and its derivatives: an important new class of antimalarial agents.” Pharmacology & therapeutics 90, no. 2-3 (2001): 261-265.


10. Van Agtmael, Michiel A., Teunis A. Eggelte, and Chris J. van Boxtel. “Artemisinin drugs in the treatment of malaria: from medicinal herb to registered medication.” Trends in Pharmacological Sciences 20, no. 5 (1999): 199-205.


11. De Ridder, Sanne, Frank Van der Kooy, and Robert Verpoorte. “Artemisia annua as a self-reliant treatment for malaria in developing countries.” Journal of ethnopharmacology 120, no. 3 (2008): 302-314.


12. Delabays, N., Simonnet, X., Gaudin, M., 2001. The genetics of artemisinin content in Artemisia annua L. and the breeding of high yielding cultivars. Current Medicinal Chemistry 8, 1795–1801.


13. Hsu, E., 2006. The history of Qing Hao in the Chinese Materia medica. Transactions of the Royal Society of Tropical Medicine and Hygiene 100, 505–508.


14. De Ridder, Sanne, Frank Van der Kooy, and Robert Verpoorte. “Artemisia annua as a self-reliant treatment for malaria in developing countries.” Journal of ethnopharmacology 120, no. 3 (2008): 302-314.


15. Fakhreldin M. Omer, J. Brian de Souza, Eleanor M. Riley. Differential Induction of TGF-{beta} Regulates Proinflammatory Cytokine Production and Determines the Outcome of Lethal and Nonlethal Plasmodium yoelii Infections. J. Immunol. 2003;171;5430-5436.


16. Claire L. Mackintosh, James G. Beeson, Kevin Marsh. Clinical features and pathogenesis of severe malaria. Trends in Parasitology December 2004;20(12):597-603.


17. Ian A Clark, Alison C Budd, Lisa M Alleva, William B Cowden. Human malarial disease: a consequence of inflammatory cytokine release. Malaria Journal. 2006;5:85.


18. Cumming, Jared N., Poonsakdi Ploypradith, and Gary H. Posner. “Antimalarial activity of artemisinin (qinghaosu) and related trioxanes: mechanism (s) of action.” In Advances in pharmacology, vol. 37, pp. 253-297. Academic Press, 1996.


19. Posner, Gary H., and Paul M. O’Neill. “Knowledge of the proposed chemical mechanism of action and cytochrome P450 metabolism of antimalarial trioxanes like artemisinin allows rational design of new antimalarial peroxides.” Accounts of chemical research 37, no. 6 (2004): 397-404.


20. O’neill, Paul M., Victoria E. Barton, and Stephen A. Ward. “The molecular mechanism of action of artemisinin—the debate continues.” Molecules 15, no. 3 (2010): 1705-1721.


21. Pai-Dhungat, J. V. “Caventou, Pelletier &-History Of Quinine.” Journal of the Association of Physicians of India 63 (2015).


22. Chang, Zengyi. “The discovery of Qinghaosu (artemisinin) as an effective anti-malaria drug: a unique China story.” Science China Life Sciences 59, no. 1 (2016): 81-88.


23. Keyaerts, Els, Leen Vijgen, Piet Maes, Johan Neyts, and Marc Van Ranst. “In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine.” Biochemical and biophysical research communications 323, no. 1 (2004): 264-268.


24. Vincent, Martin J., Eric Bergeron, Suzanne Benjannet, Bobbie R. Erickson, Pierre E. Rollin, Thomas G. Ksiazek, Nabil G. Seidah, and Stuart T. Nichol. “Chloroquine is a potent inhibitor of SARS coronavirus infection and spread.” Virology journal 2, no. 1 (2005): 1-10.


25. De Wilde, Adriaan H., Dirk Jochmans, Clara C. Posthuma, Jessika C. Zevenhoven-Dobbe, Stefan Van Nieuwkoop, Theo M. Bestebroer, Bernadette G. Van Den Hoogen, Johan Neyts, and Eric J. Snijder. “Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture.” Antimicrobial agents and chemotherapy 58, no. 8 (2014): 4875-4884.


26. Al-Bari MAA. Targeting endosomal acidification by Chloroquine analogs as a promising strategy for the treatment of emerging viral diseases. Pharmacol Res Perspect 2017, 5: e00293


27. Yan Y, Zou Z, Sun Y, et al. Anti-malaria drug Chloroquine is highly effective in treating Avian Influenza A H5N1 virus infection in an animal model. Cell Res 2013, 23: 300-2.


28. Wang, Manli, Ruiyuan Cao, Leike Zhang, Xinglou Yang, Jia Liu, Mingyue Xu, Zhengli Shi, Zhihong Hu, Wu Zhong, and Gengfu Xiao. “Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro.” Cell research 30, no. 3 (2020): 269-271.


29. Gao, Jianjun, Zhenxue Tian, and Xu Yang. “Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies.” Bioscience trends (2020).


30. Gautret, Philippe, Jean-Christophe Lagier, Philippe Parola, Line Meddeb, Morgane Mailhe, Barbara Doudier, Johan Courjon et al. “Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial.” International journal of antimicrobial agents (2020): 105949.


31. Paeshuyse, Jan, Lotte Coelmont, Inge Vliegen, Jan Vandenkerckhove, Eric Peys, Benedikt Sas, Erik De Clercq, and Johan Neyts. “Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin.” Biochemical and biophysical research communications 348, no. 1 (2006): 139-144.


32. Romero, Marta R., Thomas Efferth, Maria A. Serrano, Beatriz Castaño, Rocio IR Macias, Oscar Briz, and Jose JG Marin. “Effect of artemisinin/artesunate as inhibitors of hepatitis B virus production in an “in vitro” replicative system.” Antiviral research 68, no. 2 (2005): 75-83.


33. Efferth, Thomas, Manfred Marschall, Xin Wang, Shu-Mei Huong, Ilona Hauber, Armin Olbrich, Martina Kronschnabl, Thomas Stamminger, and Eng-Shang Huang. “Antiviral activity of artesunate towards wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses.” Journal of molecular medicine 80, no. 4 (2002): 233-242.


34. Kaptein, Suzanne JF, Thomas Efferth, Martina Leis, Sabine Rechter, Sabrina Auerochs, Martina Kalmer, Cathrien A. Bruggeman, Cornelis Vink, Thomas Stamminger, and Manfred Marschall. “The anti-malaria drug artesunate inhibits replication of cytomegalovirus in vitro and in vivo.” Antiviral research 69, no. 2 (2006): 60-69.


35. Naesens, Lieve, Pascale Bonnafous, Henri Agut, and Erik De Clercq. “Antiviral activity of diverse classes of broad-acting agents and natural compounds in HHV-6-infected lymphoblasts.” Journal of clinical virology 37 (2006): S69-S75.


36. Naesens, Lieve, Pascale Bonnafous, Henri Agut, and Erik De Clercq. “Antiviral activity of diverse classes of broad-acting agents and natural compounds in HHV-6-infected lymphoblasts.” Journal of clinical virology 37 (2006): S69-S75.


37. Efferth, Thomas, Manfred Marschall, Xin Wang, Shu-Mei Huong, Ilona Hauber, Armin Olbrich, Martina Kronschnabl, Thomas Stamminger, and Eng-Shang Huang. “Antiviral activity of artesunate towards wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses.” Journal of molecular medicine 80, no. 4 (2002): 233-242.


38. Qian, R. S., Z. L. Li, J. L. Yu, and D. J. Ma. “The immunologic and antiviral effect of qinghaosu.” Journal of traditional Chinese medicine= Chung i tsa chih ying wen pan 2, no. 4 (1982): 271.


39. Romero, Marta R., Maria A. Serrano, Marta Vallejo, Thomas Efferth, Marcelino Alvarez, and Jose JG Marin. “Antiviral effect of artemisinin from Artemisia annua against a model member of the Flaviviridae family, the bovine viral diarrhoea virus (BVDV).” Planta medica 72, no. 13 (2006): 1169-1174.


40. Lin, L., Han, Y., & Yang, Z. M. (2003). Clinical observation on 103 patients of severe acute respiratory syndrome treated by integrative traditional Chinese and Western Medicine. Zhongguo Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi= Chinese journal of integrated traditional and Western medicine, 23(6), 409.


41. Suryanarayana, Lakavath, and Dhanalaxmi Banavath. “A Review On Identification of Antiviral Potential Medicinal Plant Compounds Against with COVID-19.” International Journal of Research in Engineering, Science and Management 3, no. 3 (2020): 675-679.


42. Gilmore, K.; Osterrieder, K.; Seeberger, P.H. (2020): “Artemisia annua Plant Extracts are Active Against SARS-CoV-2 In Vitro”, in: submitted for publication


43. Marchand, Els, Magnus A. Atemnkeng, Stijn Vanermen, and Jacqueline Plaizier‐Vercammen. “Development and validation of a simple thin layer chromatographic method for the analysis of artemisinin in Artemisia annua L. plant extracts.” Biomedical chromatography 22, no. 5 (2008): 454-459.


44. Lubbe, Andrea, Isabell Seibert, Thomas Klimkait, and Frank Van der Kooy. “Ethnopharmacology in overdrive: the remarkable anti-HIV activity of Artemisia annua.” Journal of ethnopharmacology 141, no. 3 (2012): 854-859.


45. van der Kooy, Frank, and Robert Verpoorte. “The content of artemisinin in the Artemisia annua tea infusion.” Planta Medica-Natural Products and MedicinalPlant Research 77, no. 15 (2011): 1754.


46. Carbonara, Teresa, Rossana Pascale, Maria Pia Argentieri, Paride Papadia, Francesco Paolo Fanizzi, Luciano Villanova, and Pinarosa Avato. “Phytochemical analysis of a herbal tea from Artemisia annua L.” Journal of Pharmaceutical and Biomedical Analysis 62 (2012): 79-86.


47. Weathers, Pamela J., and Melissa J. Towler. “The flavonoids casticin and artemetin are poorly extracted and are unstable in an Artemisia annua tea infusion.” Planta medica 78, no. 10 (2012): 1024.


48. Xing J, Kirby BJ, Whittington D, et al. Evaluation of P450 inhibition and induction by artemisinin antimalarials in human liver microsomes and primary human hepatocytes. Drug Metab Dispos. 2012 Sep;40(9):1757-64.


49. Burk, O., Arnold, K.A., Nussler, A.K., Schaeffeler, E., Efimova, E., Avery, B.A., Avery, M.A., Fromm, M.F., Eichelbaum, M., 2005. Antimalarial artemisinin drugs induce cytochrome P450 and MDR1 expression by activation of xenosensors pregnane X receptor and constitutive androstane receptor. Molecular Pharmacology 67, 1954–1965.


50. Svensson, Ulrika SH, and M. Ashton. “Identification of the human cytochrome P450 enzymes involved in the in vitro metabolism of artemisinin.” British journal of clinical pharmacology 48, no. 4 (1999): 528.


51. Meshnick, Steven R. “Artemisinin: mechanisms of action, resistance and toxicity.” International journal for parasitology 32, no. 13 (2002): 1655-1660.


52. Zhao, K., Song, Z., 1990. The pharmacokinetics of dihydroqinghaosu given orally to rabits and dogs (chinese). Acta Pharmaceutica Sinica 25:161–163.


53. Shuhua, X., Catto, B.A., 1989. In vitro and in vivo studies of the effect of artemether on Schistosoma mansoni. Antimicrobial Agents and Chemotherapy 33:1557–1562.


54. Titulaer, H.A.C., Zuidema, J., Kager, P.A., Wetsteyn, J.C.F.M., Lugt, C.B., Merkus, F.W.H.M., 1990. The pharmacokinetics of artemisinin after oral, intramuscular and rectal administration to volunteers. J. Pharm. Pharmacol. 42:810–813.


55. Navaratman, V., Mansor, S.M., Chin, L.K., Mordi, M.N., Asokan, M., Nair, N.K., 1995. Determination of artemether and dihydroartemisinin in blood plasma by highperformance liquid chromatography for application in clinical pharmacological studies. J.Chrom. 669:289–294.

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Gain-of-Function Studies and SARS-CoV-2 Virus (COVID-19) Emergence

Gain-of-Function Studies and SARS-CoV-2 Virus (COVID-19) Emergence

Gain-of-Function Studies and SARS-CoV-2 Virus (COVID-19) Emergence

James Odell, OMD, ND, L.Ac.

Editorial – The material published in this editorial is intended to foster scholarly inquiry and rich discussion of the controversial topic of bioethics and health policy. The views expressed in this article are solely the authors and do not represent the policy or position of the Bioregulatory Medicine Institute (BRMI), nor any of its Board Advisors or contributors. The views expressed are not intended to malign any religious or ethnic group, organization, company, individual, or any other.Every effort has been made to attribute the sources of this article to the rightful authors

Gain-of-function (GOF) research involves experimentation that aims to (and actually does) increase the transmissibility and/or virulence of pathogenic viruses. GOF research typically involves mutations that confer altered functionality of a protein, molecule, or organism such as a virus. Such research (when safely conducted by responsible scientists) allegedly intends to improve the understanding of disease-causing agents, their interaction with human hosts, and/or even their potential to cause pandemics. But concerns about the safety of GOF studies have been voiced by numerous scientists from their very beginning. No matter how anyone justifies it or spins it, GOF studies manipulate pathogenic, deadly viruses to increase their transmissibility or virulence. Within the field of virology, these studies create ‘chimeric viruses’ defined as a new hybrid microorganism.  These are created by joining nucleic acid fragments, from two or more different microorganisms, in which each of at least two of the fragments contains essential genes necessary for replication.  This type of research has proven to be historically unsafe. Serious questions arise as to whether GOF research and the development of chimeric viruses are necessary for understanding viruses considering its potential for deadly contamination (pandemics) and bioterrorism. 

The term “potential pandemic pathogen (PPP)” was coined for such manufactured viruses. The first suspected experimental effort to create a PPP occurred in 2005 with a laboratory re-creation of a strain of influenza, H1N1 from 1918, by Tumpey and colleagues. This was based on synthesizing nucleic acid sequences obtained from partially preserved viral RNA in frozen corpses from 1918, and then creating infectious viruses by reverse genetics. The pandemic influenza virus of 1918–1919 killed an estimated 20 to 50 million people worldwide. The question was raised as to whether it was wise to construct a virus that was historically associated with the worst pandemic in modern history and somewhat different from any virus currently circulating. However, the debate seems to have been internal to the US Department of Health and Human Services (HHS), particularly the National Institutes of Health (NIH), and it was judged that the work should proceed.

In the United States, more GOF study controversy occurred in 2011 when two laboratories published reports of mutational screens of H5N1 avian influenza viruses, identifying variants which proved transmissible through the air between ferrets. The Dutch virologist Ron Fouchier, based at Erasmus Medical Center in Rotterdam, and Yoshihiro Kawaoka at the University of Wisconsin-Madison announced that they had successfully mutated H5N1, a strain of bird flu, to pass through the air between ferrets, in two separate experiments. Ferrets are considered one of the best flu models because their respiratory systems react to the flu much like humans. The mutations that gave the virus its ability to be airborne transmissible were gain-of-function mutations. Of course, the concern then became that if their mutated H5N1 ever left the lab it could cause a pandemic.

Around this time more scientists raised concerns about the potential of a laboratory accident that could lead to the release of a pathogen that, by design, combined high virulence and antigenic novelty with high human-to-human transmissibility. These early critiques also questioned whether the scientific and public health value of such research justified the risks involved. One of the first major discussion meetings on this topic, to my knowledge, was held at the Royal Society of London in 2012 (, with mainly UK and North American speakers.

In 2012 and 2013, contamination and safety concerns continued to be published throughout the media and voiced by the international scientists.1 Then in 2014, as many as 75 scientists at the Center of Disease Control and Prevention were exposed to anthrax. A few weeks later, FDA officials discovered 16 forgotten vials of smallpox in storage. Meanwhile, the largest, most severe, and most complex Ebola outbreak in history was raging across West Africa, and the first patient to be diagnosed in the US had just been announced. On October 16, 2014 following these biosafety mishaps involving anthrax, smallpox, and H5N1 in government laboratories, the White House Office of Science and Technology Policy announced the launch of the U.S. Government in implementing a deliberative process to re-evaluate the potential risks and benefits associated with certain GOF experiments. The then Obama administration paused the release of federal funding for GOF studies anticipated to enhance the pathogenicity or transmissibility of respiratory influenza droplets among mammals and, in particular the COVID viruses – SARS and MERS. Thus, there was a moratorium on GOF between 2014 and 2017on the grounds of safety concerns and views of many top scientists who considered this type of viral research ‘unnecessarily dangerous’ – with potential risks of accidental release of pandemic potential viruses.

Following the funding pause, two major discussion meetings were held by the US National Academy of Sciences, and multiple meetings were held by the NSABB. A few ethicists, and others, debated the scientific and public health rationale for GOF or PPP experiments, the risks they posed, and the ethics of performing research that poses potentially major risks to unaware persons not involved in the studies. This process raised awareness of many issues that had not been previously highlighted, notably the lack of a framework for assessing research risks to persons who are not research participants, the very poor availability of data on biosafety in biological laboratories in the US and elsewhere, and the consequent uncertainty in risk-benefit calculations.

GOF Research in Europe

The debate on the risks and merits of GOF research has not been limited to the United States, as the Dutch Court of Appeals recently handed down a verdict concerning Erasmus University Medical Centers (EMC) objection to export license rules regarding the publication of highly pathogenic avian influenza virus GOF research. Export licenses are in place in the European Union to prevent the proliferation of weapons of mass destruction and apply to specific biological agents, chemical agents, and technologies. In 2012, the Dutch government ruled that EMC had to apply for an export license to publish their GOF work, which they had done in order to expedite publication. However, EMC later filed an objection, maintaining that GOF research in this context was for “basic scientific research.” The Dutch Court of Appeals ruled that EMC had no legal standing to contest the export license regulations, but did not address the legality of the export license itself, leaving the issue open for further debate. Currently, all GOF research within the European Union requires an export license for publication.

Laboratory Mishaps

GOF laboratory contamination mishaps have historically occurred on more than one occasion. In 1975 smallpox, the deadly infectious disease that killed about 30 percent of those who contracted it, finally became eradicated from the world. Around 300 million-plus people died of smallpox in the century before it was annihilated. In 1978, smallpox suddenly appeared again in Birmingham, England when Janet Parker, a photographer at Birmingham Medical School developed a horrifying rash. 2 The doctors initially mistakenly diagnosed it as chickenpox. Parker’s condition worsened and she was admitted to the hospital, where testing determined she had smallpox. Unfortunately, she died of the disease a few weeks later. People then questioned how she acquired smallpox that was supposed to have been eradicated. It turned out that the building Parker worked in also contained a research laboratory, one of a handful in the world where smallpox was still studied. Some papers reported the lab was badly mismanaged 3, with important safety precautions being ignored. Interestingly, the doctor who ran the lab died by alleged suicide shortly after Parker was diagnosed. So somehow, smallpox escaped the lab to infect this individual elsewhere in the building. Through sheer luck and a rapid response from health authorities, including quarantine of more than 300 people, the deadly error did not turn into an outright pandemic.

As previously mentioned, in 2014 the FDA did a cleanup for a planned move to a new office. They found hundreds of unclaimed vials of virus samples in a cardboard box in the corner of a cold storage room.4 Six of them, it turned out, were vials of smallpox. No one had been keeping track of them and no one apparently even knew they were there. They may have been there since the 1960s. The surprised and panicked scientists put the materials in a box, sealed it with clear packaging tape, and carried it to a supervisor’s office. This of course is not approved handling of dangerous biological materials. It was later found that the integrity of one vial was compromised, luckily, not one containing a ‘deadly’ virus. Additionally, there is also strong circumstantial evidence that the reintroduction of H1N1 into human circulation in 1977 after its disappearance in 1950 began with the accidental release of a laboratory strain.5, 6

Lab mishaps continue to occur and with GOF studies creating more virulent and pathogenic organisms, it becomes only a matter of time before dangerous organisms escape into the world or are used in bioterrorism. Highly transmissible, highly virulent GOF viruses like the modified H5N1 strains that have been created have the ability to infect millions and potentially kill a large fraction of those afflicted.

Current GOF Risks and Policy

GOF risks fall into two general categories which are separate but related: namely, biosecurity and biosafety. Biosecurity risk is the likelihood that someone would use the products or information obtained regarding a more pathogenic virus from GOF experiments that led to a more pathogenic virus that caused intentional damage in the form of bioterrorism. Biosafety risk is the likelihood of accidental escape that could trigger an outbreak and epidemic.

Here is a more in-depth review of the risk of GOF studies:

• Biosafety—i.e. health dangers associated with laboratory accidents

• Biosecurity—i.e., health dangers associated with crime and terrorism if pathogens are not physically secure and/or if malevolent actors gain access to them.

• Proliferation—i.e., dangers that might grow proportionally with an increased rate of GOF, potentially in different settings with varying biosafety standards.

• Information risk—i.e., if published studies facilitate malevolent action (e.g., by terrorists) or, possibly, breach of intellectual property.

• Agricultural—i.e., risks to agriculturally-relevant animals if enhanced pathogens arising from GOF are accidentally or intentionally released into animal populations, and possible implications for human health.

• Economic risks—i.e., financial implications of (accidental or intentional) pathogen release with resulting stock market collapse, business bankruptcies, job losses with increasing unemployment, starvation increases, suicides, and overall health-care downfall.

• Loss of public confidence—i.e., compromise of trust (in the scientific enterprise) that could result from (accidental or intentional) pathogen release.

Resuming Gain-of Function Studies

On Dec 19, 2017, the US National Institutes of Health (NIH) announced that they would resume funding GOF experiments involving influenza, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus. This ended the safety moratorium.

In review, during the GOF moratorium, a panel called the National Science Advisory Board for Biosecurity spent months designing a new process for determining the risks and benefits of GOF studies that could make pathogens more likely to spread and cause serious disease in humans. That led to a December 2017 HHS review framework for research on what the government now calls enhanced potential pandemic pathogens (enhanced PPPs). The policy stipulates that after a proposed enhanced PPP experiment passes NIH scientific peer review, an HHS panel of federal officials with wide-ranging expertise weighs the risks and benefits. If the committee approves, it can then receive NIH funding.

Then in February 2019, the magazine Science reported that the HHS review panel had approved two H5N1 projects in labs in Wisconsin and the Netherlands.7 These approvals and funding were for the same labs (Kawaoka and Fouchier labs) that created the controversy in 2011. Remembering that in 2011, Fouchier and Kawaoka alarmed the world by revealing they had separately modified the deadly avian H5N1 influenza virus so that it spread between ferrets.8 The news greatly disturbed opponents of such research, and they criticized federal officials for not disclosing the approvals in an op-ed in The Washington Post.9 HHS and NIH soon publicized the two approved projects, but did not release the risk reviews.

Today, much of this GOF research has little to zero transparency, particularly what is funded by the NIH and conducted in China at the Wuhan Institute of Virology. This P4 lab in Wuhan (P4 is an exceedingly high biosafety level designation) is not only the first of its kind in China, but also the first in Asia. When it opened in 2017, U.S. scientists expressed concerns that, considering China’s opaque administrative structure, if one of those killer viruses “escaped” from the lab, it could cause a doomsday disaster.

Wuhan Institute of Virology, a biosafety level 4 laboratory located in Jiangxia District, Wuhan that has engaged in gain-of-function research.

Hector Retamal/AFP/Getty Images

Dr. Marc Lipsitch is Professor of Epidemiology and Director of the Center for Communicable Disease Dynamics at the Harvard School of Public Health. He is an author of more than 100 peer-reviewed publications on antimicrobial resistance, mathematical modeling of infectious disease transmission, bacterial and human population genetics, and immunity to Streptococcus pneumoniae. Dr. Lipsitch was quoted,7 “I still do not believe a compelling argument has been made for why these studies (GOF) are necessary from a public health point-of-view; all we have heard is that there are certain narrow scientific questions that you can ask only with dangerous experiments”, he said. “I would hope that when each HHS review is performed someone will make the case that strains are all different, and we can learn a lot about dangerous strains without making them transmissible.” Lipsitch pointed out that every mutation that has been highlighted as important by a gain-of-function experiment has been previously highlighted by completely safe studies. “There is nothing for the purposes of surveillance that we did not already know”, said Dr. Lipsitch. “Enhancing potential pandemic pathogens in this manner is simply not worth the risk.”10

Gain-of-Function – Chimera Virus Research in China

Since the SARS virus of 2003 China launched extensive virology research programs. These occurred through their military labs (People’s Liberation Army) and other labs such as the P4 virology lab in Wuhan located, apparently, just 280 meters away from the Hunan Seafood Market. The P4 lab in Wuhan was initially started as a joint venture with the French government. Chinese authorities switched the management of the project to a firm with close ties to the Chinese military complex. The Lab is a subsidiary of the Wuhan Institute of Virology managed by the China Academy of Sciences. The Chinese government has been working on GOF coronavirus studies for well over a decade. One of the most renowned Chinese virologists of this field is Shi Zhen-Li (surname Shi 石) who is renowned for her extensive research of SARS-like coronaviruses of bat origin. Since the SARS virus outbreak in 2003, Shi Zhengli and her team have conducted research on coronaviruses. In 2005, Shi and colleagues found that bats are a natural reservoir of SARS-like coronaviruses.11 To further determine the mechanism by which a SARS-associated coronavirus (SARS-CoV) may infect humans, Shi led a research team that studied the binding of spike proteins (s-protein) of both natural and chimeric SARS-like coronaviruses to ACE2 receptors in human, civet, and horseshoe bat cells.12, 13 ACE2’s functions include ultimately acting as a vasodilator that influences blood flow. It is located on cells all around the body, but ACE2 receptors also occur in many organs. It is especially common on cells lining air sacs (pneumocytes) in the lungs, which is partly why infection is associated with respiratory symptoms like pneumonia.

From 2010 onwards, Shi and her team have been primarily focused on identifying the capacity for coronavirus transmission across species, specifically putting the emphasis of study on the s-protein, or spike protein, of the coronaviruses. To successfully initiate an infection, viruses need to overcome the cell membrane barrier. Enveloped viruses achieve this by membrane fusion, a process mediated by specialized viral fusion proteins.

For coronaviruses, this fusion occurs through its spike protein. Each spike protein consists of three components that combine to form a ‘trimer’ structure with two parts or ‘subunits’, S1, and S2. You can think of the spike as a multistage rocket, with S1 being the boosters and S2 as a space shuttle: once attached to the ACE2 receptor, a spike sheds its S1 subunit and the remaining S2 part changes its shape or ‘conformation’ to enable the viral envelope to fuse with the outer membrane and drop the virus’ genetic material inside the cell.

The spike proteins (shown sticking out from the round virus) have high homology with the SARS virus. These are the proteins that make up the “key” that binds with the ACE2 receptors in humans to enter the cell. This contributes to the organ failure we see with infected persons as it drives the virus into the cells of the lungs and other organs such as the heart and kidneys, which also have ACE2 receptors.

Coronavirus​ / CC BY-SA

Thus, for SARS-CoV entry into a host cell, its s-protein needs to be cleaved by cellular proteases at 2 sites, termed S protein priming, so the viral and cellular membranes can fuse.14 In other words, Shi and her research team has been dedicated to finding ways that can better allow bat coronavirus to be transmissible to other animals.

In June of 2010, Shi’s team published a paper that describes research to understand the susceptibility of angiotensin-converting enzyme 2 (ACE2) proteins of different bat species to the s-protein of the SARS virus.15 With their chimeric research, they also modified key amino acid components to mutate the bats’ ACE2 receptor in order to examine compatibility with the SARS s-protein. This paper demonstrated their awareness of the relationship between the s-protein and the ACE2 receptor.

It is now understood that 2019-nCoV can infect the human respiratory epithelial cells through interaction with the human ACE2 receptor. Indeed, the recombinant spike protein can bind with the recombinant ACE2 protein. Shi and her colleagues’ paper also signified that they had discovered the passageway for coronaviruses to infect human bodies.

Angiotensin-converting enzyme 2, a monocarboxylase that degrades angiotensin II to angiotensin 1–7, is also the functional receptor for severe acute respiratory syndrome (SARS) coronavirus (SARS‐CoV) and is highly expressed in the lungs and heart. Patients with SARS also suffered from cardiac disease including arrhythmias, sudden cardiac death, and blood pressure -systolic and diastolic dysfunction.

In 2013, Shi and her team published a paper in the journal Science China Life Sciences in which they isolated and identified numerous bat viruses (bat lyssaviruses, bat paramyxoviruses, bat filoviruses, bat reoviruses, and others).Bats are the only mammals capable of sustained flight and are notorious ‘reservoir hosts’ for some of the world’s most highly pathogenic viruses, including Nipah, Hendra, Ebola, and SARS.16 A reservoir is one or several animal species that are not or not very sensitive to the virus, which will naturally host one or several viruses. The absence of symptoms of the disease is explained by the effectiveness of their immune system, which allows them to fight against too much viral proliferation.

Bats Have Been Linked with Seven Major Epidemics Over the Past Three Decades.

In October 2013 Shi and her team published their findings in the prestigious journal Nature and claimed a breakthrough in coronavirus research.17 They provide evidence that SARS-CoV originated in bats. They concluded, “Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs.” In their research, they isolated three bat viruses, one of which had an s-protein that integrated with human ACE2 receptors. This effectively demonstrated the direct human infection of SARS-like viruses to humans without the need for an intermediate host. Then in 2014, Shi and her team collaborated on additional gain-of-function experiments led by Ralph S. Baric of the University of North Carolina, which showed that two critical mutations that the MERS coronavirus possesses allow it to bind to the human ACE2 receptor.18 In 2015, She and colleagues further showed that SARS had the potential to re-emerge from coronaviruses circulating in bat populations in the wild.19

Then in November 2015, Shi and her team from Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology together with scientists from the Department of Epidemiology at the University of North Carolina at Chapel Hill, published a paper in the journal Nature Medicine describing their joint gain of function coronaviruses research and new chimeric virus creation. According to Ralph Baric, an infectious-disease researcher at the University of North Carolina at Chapel Hill and co-author of the study, this study began before the US moratorium was enacted. So, the US National Institutes of Health (NIH) allowed it to proceed while it was under review by the agency. Baric claims the NIH eventually concluded that the work was not ‘so risky’ as to fall under the moratorium.20

They revealed the formation of a new synthetic virus or self-replicating chimeric virus. This chimeric virus had SARS virus as the framework with the key s-protein replaced by one they had found in a coronavirus, she mentioned in her 2013 paper. They concluded “we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo (mice).” In short, they took genes from a bat coronavirus spike-protein and spliced it to a mouse coronavirus genome, then tested this for its ability to infect human airway cells through their ACE-2 receptors. This chimeric recombinant coronavirus was tested in mice with significant deadly infections occurring. According to their paper, “all mouse studies were performed at the University of North Carolina, prior to the 2014 GOF moratorium involving influenza, MERS and SARS viruses.” This new virus demonstrated a powerful ability for cross species infection. The mice infected with this chimeric virus showed severe lung damage with no cure. Shi Zhengli’s team and her US colleagues’ successful recombinant splicing of the SARS virus was strategically important to the development of cross-species transmission. Their study eerily concluded that there was “a significant risk of a SARS coronavirus re-emergence”.

The fact that scientists are deliberately manipulating the genetics of deadly viruses to manufacture chimeric viruses and then test them for their ability to cause human disease should have created an outrage in the scientific community. This event was not even reported in mainstream media. The medium (chimeric coronavirus) from transfected cells was harvested and served as seed stocks for subsequent experiments (to be performed later).

An additional piece of vaccination information emerged from their 2015 study entitled ‘A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence’. They revealed that in order “to evaluate the efficacy of existing vaccines against infection with SHC014-MA15 (chimeric coronavirus), we vaccinated aged mice with double-inactivated whole SARS-CoV (DIV). Previous work showed that DIV could neutralize and protect young mice from challenge with a homologous virus; however, the vaccine failed to protect aged animals in which augmented immune pathology was also observed, indicating the possibility of the animals being harmed because of the vaccination. Here we found that DIV did not provide protection from challenge with SHC014-MA15 with regards to weight loss or viral titer. Consistent with a previous report with other heterologous groups 2b CoVs, serum from DIV-vaccinated, aged mice also failed to neutralize SHC014-MA15. Notably, DIV vaccination resulted in robust immune pathology and eosinophilia). Together, these results confirm that the DIV vaccine would not be protective against infection with SHC014 and could possibly augment disease in the aged vaccinated group.”21 The phenomena that specific viral vaccines can exacerbate an existing viral disease is well documented, particularly with influenza vaccines.22, 23, 24, 25, 26, 27

In summary, the researchers created and examined the disease potential of a synthetic SARS-like virus, SHC014-MA15, which was chimerically created from viruses found in Chinese horseshoe bat populations. Using what they described as “SARS-CoV reverse genetics system”, the researchers said that they generated and characterized a “chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone.” The mice died a grim death and the experiment was hailed a success by the researchers.

Back in Wuhan, Shi and her team then focused their chimeric viral research on primates. At this stage, some scientists took notice and became genuinely concerned, understanding this development was a dangerous move towards simulating the infection in humans. Academic debates on GOF studies one again began worldwide.

Dr. Wain-Hobson of the Pasture Institute in France expressed his disapproval and concern. In an article, he told Nature, “If the virus escaped, nobody could predict the trajectory.” Richard Ebright, a molecular biologist and biodefence expert at Rutgers University in Piscataway, New Jersey agrees and said, “The only impact of this work is the creation, in a lab, of a new, non-natural risk.”28 Both Drs Ebright and Wain-Hobson are long-standing critics of GOF research.

In October of 2014, the Obama Administration wary of the potential threats to public health and GOF research suspended funding to these research projects. Funding cuts and scientific criticism of GOF studies however did not stop Shi Zhengli’s team research. She and her colleagues continued under the cover and funding of the Chinese Government.

The question then is why would anyone be creating a virulent designer coronavirus that can infect humans? For what purpose is this research? Is it for a bioweapon? Is it so that you can create a vaccine and be the recipient of the profits? The standard pro-gain-of-function narrative is that the ultimate objective of such research is to better inform public health and preparedness efforts and/or development of medical countermeasures. Many scientists disagree. As Dr. Marc Lipsitch pointed out, every mutation that has been highlighted as important by a gain-of-function experiment has been previously brought to light by completely safe studies.

NIH Funding Resumes

On Dec 19, 2017, the NIH announced that they would again resume funding gain-of-function experiments involving influenza, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus ending the safety moratorium.29 This once again opened the door to NIH funding to GOF studies China. As a safety condition, the federal government then began requiring that any National Institutes of Health grant proposals involving gain-of-function research undergoes a review by an expert panel to evaluate the risk of such work against the potential gains. But the names of the expert-panel members are not publicly available, nor are its reviews of study proposals. “We’re not trying to say the policy is wrong, we’re trying to say the policy is ambiguous,” says Dr. Marc Lipsitch, an epidemiologist at the Harvard T.H. Chan School of Public Health in Boston, Massachusetts, and one of the researchers calling for greater transparency around such work.

In 2019, the NIH committed $3.7 million over six years for research on bat coronaviruses in China. The program followed a previous $3.7 million, a 5-year project for collecting and studying bat coronaviruses, which ended in 2019, bringing the total to $7.4 million. One primary concern by many scientists is some of this newest additional 3.7 million was directed to the Wuhan Institute of Virology for coronavirus GOF studies. When the NIH was questioned about the grants they responded, “The grant you are referencing is a multi-site, multi-country project supporting research that aims to understand what factors allow coronaviruses, including close relatives to SARS, to evolve and jump into the human population and cause disease. Specifically, the project includes studying viral diversity in animal (bats) reservoirs, surveying people that live in high-risk communities for evidence of bat-coronavirus infection, and conducting laboratory experiments to analyze and predict which newly-discovered viruses pose the greatest threat to human health.”30 The National Institute of Allergy and Infectious Diseases (NIAID), under Anthony Fauci’s leadership, has financially supported six studies of bats and their connection to coronavirus.

SARS-CoV-2 Origin Debate

Many rumors and debates have persisted as to the origins of the SARS-CoV-2 virus. Most claim it came from bats and jumped into humans, acquiring new genomic features through adaptation during undetected human-to-human transmission. The official narrative from the WHO and the CDC is that the SARS-CoV-2 virus was a natural development or mutation, from a still-unknown animal source. An excerpt from the website states, “The SARS-CoV-2 virus is a betacoronavirus, like MERS-CoV and SARS-CoV-1. All three of these viruses have their origins in bats. The sequences from U.S. patients are similar to the one that China initially posted, suggesting a likely single, recent emergence of this virus from an animal reservoir. Early on, many of the patients at the epicenter of the outbreak in Wuhan, Hubei Province, China had some link to a large seafood and live animal market, suggesting animal-to-person spread. Later, a growing number of patients reportedly did not have exposure to animal markets, indicating person-to-person spread.”

The Chinese government from the beginning has claimed the virus originated in the Hunan Seafood Market in Wuhan in December 2019. This was propagated by mainstream media and at first, was believable because 27 of the first 41 people hospitalized (66 percent) passed through a market located in the heart of Wuhan city. Yet, later it was discovered that there is also genomic evidence and reports of the virus having circulated earlier in November. A molecular dating estimate based on the SARS-CoV-2 genomic sequences indicate an origin in November 2019.31 They also concluded, “ that the human SARS‐CoV‐2 virus, which is responsible for the current outbreak of COVID‐19, did not come directly from pangolins.” Interestingly, bats were not sold in the Hunan Seafood Market. This raises questions about the link between this COVID-19 epidemic and wildlife in the Hunan Seafood Market.

Thus, the Hunan Seafood Market origin scenario is highly unlikely, particularly now that other evidence exists of numerous individuals before December with the virus who had no contact with anyone from the market. Theories about laboratory contamination surfaced immediately in China, primarily because of the proximity of the Hunan Seafood Market from the Wuhan Institute of Virology. Particularly with the ongoing history of chimeric bat coronaviruses being studied and created there.

The Chinese government has strongly pushed back conjecture that the virus was lab leaked or lab manufactured. Wuhan Institute of Virology director Wang Yanyi recently told state broadcaster CGTN that the theory Covid-19 leaked from a lab in Wuhan is “pure fabrication.” Wang adds “her lab was studying three live strains of bat coronaviruses, but none matched Covid-19.”32 An analysis by a team from the Wuhan Institute of Virology, posted to the preprint server bioRxiv, claimed that the genome of this coronavirus (the seventh known to infect humans) is 96% identical to that of a bat coronavirus, suggesting that species is the original source.33

A recent study was published in the Nature Medicine journal34 in which the authors investigated the genetic code of a key part of the coronavirus and compared this to other known coronaviruses. They concluded that “Human-SARS CoV-2 was a natural mutation from one of several possible animal sources, of which still has not been identified.” The figure below from this study demonstrates the genetic code differences between the different animal coronavirus types and the 2002 SARS coronavirus as well. The marked and different colored areas show genetic differences.

The foremost problem with the scientists’ conclusion is the insertion of a 12-nucleotide section in the “Human-SARS CoV-2” coronavirus sequence which is completely missing from every other coronavirus type known. Such a large genetic difference (insertion) does not suddenly happen at random or naturally. This sequence was not even present in the alleged bat coronavirus as the source of this pandemic. Thus, this insertion strongly suggests that this Human-SARS CoV-2 was manufactured in a lab. The researchers claim the functional consequence of this inserted sequence (polybasic cleavage site) is unknown, but that it appears to enhance infection in human cells. They still contend that this virus originating from a laboratory is “improbable” – that means possible.

They stated,

“Polybasic cleavage sites have not been observed in related ‘lineage B’ beta coronaviruses, although other human beta coronaviruses, including HKU1 (lineage A), have those sites, and predicted O-linked glycans. Given the level of genetic variation in the spike, it is likely that SARS-CoV-2-like viruses with partial or full polybasic cleavage sites will be discovered in other species. The functional consequence of the polybasic cleavage site in SARS-CoV-2 is unknown, and it will be important to determine its impact on transmissibility and pathogenesis in animal models. Experiments with SARS-CoV have shown that insertion of a furin cleavage site at the S1–S2 junction enhances cell-cell fusion without affecting viral entry.”35

a, Mutations in contact residues of the SARS-CoV-2 spike protein. The spike protein of SARS-CoV-2 (red bar at the top) was aligned against the most closely related SARS-CoV-like coronaviruses and SARS-CoV itself. Key residues in the spike protein that make contact to the ACE2 receptors are marked with blue boxes in both SARS-CoV-2 and related viruses, including SARS-CoV (Urbani strain).

b, Acquisition of polybasic cleavage site and O-linked glycans. Both the polybasic cleavage site and the three adjacent predicted O-linked glycans are unique to SARS-CoV-2 and were not previously seen in lineage B beta coronaviruses.

Studies show that the s-protein of the new SARS-CoV-2 virus, like 2002 SARS-CoV-1 counterpart binds angiotensin-converting enzyme 2 (ACE-2), but with much higher affinity and faster binding kinetics.36, 37 This finding is particularly interesting, remembering how Shi Zhengli and her team previously investigated ways their chimeric virus could better infect the ACE-2 receptors of human and animal cells.38

Recently, Dr. Luc Montagnier during a TV interview with a French TV channel stated that elements of the HIV-1 retrovirus, which he co-discovered in 1983, can be found in the genome of the new SARS-CoV-2. Along with Françoise Barré-Sinoussi and Harald Zur Hause, Luc Montagnier won the 2008 Nobel Prize for Medicine for the discovery of human immunodeficiency virus (HIV).

Dr. Montagnier said “There has been a manipulation of the virus: at least part of it, not all of it. There is one model, which is the classic virus, which comes mainly from bats, but to which HIV sequences have been added,” he said. “In any case, it’s not natural,” he continued. “It’s the work of professionals, of molecular biologists. Very meticulous work. For what purpose? I don’t know. One hypothesis is that they wanted to create an AIDS vaccine.”

To support his theory, Montagnier cited the study by a group of researchers at the Indian Institute of Technology in New Delhi titled, “Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp 120 and Gag,”

The abstract of the article read, “We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019- nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this virus.”

The study was criticized by authorities and later withdrawn by its authors. Dr. Montagnier also predicted the imminent disappearance of the virus, because its supposedly artificial origin would be weakening it.“One can do anything with nature, but if you make an artificial construction, it is unlikely to survive. Nature loves harmonious things; what is alien, like a virus coming from another virus, for example, is not well tolerated,” he said. For the scientist, the parts of the virus into which HIV was inserted are rapidly mutating, causing it to self-destruct.

In a separate podcast episode with a different outlet, Montagnier further said the virus had escaped in an “industrial accident” from the Wuhan city laboratory when Chinese scientists were attempting to develop a vaccine against HIV. Dr. Montagnier claims have since been strongly attacked by the media and his TV interview censored.

Based on the history trail of bat COVID research, and reports from microbiologists, it suspiciously ‘appears’ that the SARS CoV-2 is not a natural mutation of any known coronavirus strain but in fact, a manmade – chimeric – strain that likely escaped the Wuhan lab. Time will tell the truth.

Biosafety Laboratories on the Rise – Regulations are Questionable

According to a 2011 report by the National Research Council, an arm of the U.S. National Academy of Sciences, hundreds of BSL-3 laboratories may be unknown, because “no federal agency is required to track the number of biocontainment labs.” 39, 40 Globally, BSL-3 labs have recently been built or are under construction in Bangladesh, India, Indonesia, China, Brazil, and Mexico, among others. Yet many countries have few or no regulations, the NRC concluded. The more secure but dangerous BSL-4 labs are also proliferating. A 2011 workshop in Istanbul organized by the NRC was told that there are 24 BSL-4 facilities, including in Germany, Gabon, Sweden, Russia, South Africa, and Canada. The United States has six BSL-4 laboratories.

In 2019 the BSL-4 Army laboratory at Fort Detrick that studies deadly infectious organisms like Ebola, anthrax and smallpox was shut down for a period of time after a CDC inspection, with many projects being temporarily halted. The lab itself reported that the shutdown order was due to ongoing infrastructure issues with wastewater decontamination, and the CDC declined to provide the reason for the shutdown due to national security concerns.41

While excellent biosafety conditions in the laboratories performing GOF studies are certainly important, it is not a panacea for guaranteeing safety. Of the major mishaps at US government labs in recent years, nearly all involved removing the infectious agent from the high-containment lab where it was under study to another, lower-containment lab because it was thought to be inert. High-tech containment cannot prevent the deliberate removal of supposedly safe material from a laboratory, and so human error remains a source of potential missteps, regardless of the quality of the laboratory facilities.

GOF studies have been one of the most hotly debated science policy issues during the 21st century, with the controversy surrounding a series of published experiments with potential implications for biological weapons-making. Such studies include the genetic engineering of a superstrain of the mousepox virus in 200142, the artificial synthesis (via synthetic genomics) of a “live” poliovirus from chemical components in 200243, and the reconstruction (via synthetic genomics) of the 1918 “Spanish Flu” virus in 2005.44 Though all of these studies involved claimed legitimate aims, critics argued that they should not have been conducted and/or published. Some argued that publishing studies like these in full detail provided “recipes” for especially dangerous potential biological weapons agents to would-be bioterrorists. Whether or not COVID-19 is eventually determined to have originated from gain-of-function research, this pandemic should be a stark reminder of the dangers it poses.

Considering the recent COVID-19 pandemic, GOF studies with the potential to enhance the pathogenicity or transmissibility of potential pandemic pathogens have once again raised biosafety and biosecurity fear and apprehension. Of concern in the context of life science research is that GOF advances in biotechnology may enable the development and use of a new generation of biological weapons of mass destruction. GOF studies can add to the evidence base, but it cannot qualitatively change that evidence base. Empirically, the contribution of such studies to applied public health goals has been far more modest than claimed. Vast advances in the most essential questions of influenza virology and in the public health goal of pandemic preparedness can be achieved without undertaking experiments that, if an accident occurs, could start a new pandemic. Until more evidence is shown that GOF studies can be conducted safely, or are even necessary, it would be wise and prudent to enact another moratorium on all GOF funding and studies.


1.; 2012.




5. Webster, Robert G., William J. Bean, Owen T. Gorman, Thomas M. Chambers, and Yoshihiro Kawaoka. “Evolution and ecology of influenza A viruses.” Microbiology and molecular biology reviews 56, no. 1 (1992): 152-179.

6. Wertheim, Joel O. “The re-emergence of H1N1 influenza virus in 1977: a cautionary tale for estimating divergence times using biologically unrealistic sampling dates.” PloS one 5, no. 6 (2010).




10. Lipsitch, Marc, and Barry R. Bloom. “Rethinking biosafety in research on potential pandemic pathogens.” MBio 3, no. 5 (2012): e00360-12.

11. Li, Wendong, Zhengli Shi, Meng Yu, Wuze Ren, Craig Smith, Jonathan H. Epstein, Hanzhong Wang et al. “Bats are natural reservoirs of SARS-like coronaviruses.” Science 310, no. 5748 (2005): 676-679.

12. Yu, Meng, Mary Tachedjian, Gary Crameri, Zhengli Shi, and Lin-Fa Wang. “Identification of key amino acid residues required for horseshoe bat angiotensin-I converting enzyme 2 to function as a receptor for severe acute respiratory syndrome coronavirus.” Journal of General Virology 91, no. 7 (2010): 1708-1712.

13. Hou, Yu-xuan, Cheng Peng, Zheng-gang Han, Peng Zhou, Ji-guo Chen, and Zheng-li Shi. “Immunogenicity of the spike glycoprotein of Bat SARS-like coronavirus.” Virologica Sinica 25, no. 1 (2010): 36-44.

14. Belouzard, Sandrine, Victor C. Chu, and Gary R. Whittaker. “Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites.” Proceedings of the National Academy of Sciences 106, no. 14 (2009): 5871-5876.

15. Hou, Yuxuan, Cheng Peng, Meng Yu, Yan Li, Zhenggang Han, Fang Li, Lin-Fa Wang, and Zhengli Shi. “Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry.” Archives of virology 155, no. 10 (2010): 1563-1569.

16. Shi, ZhengLi. “Emerging infectious diseases associated with bat viruses.” Science China Life Sciences 56, no. 8 (2013): 678-682.

17. Ge, Xing-Yi, Jia-Lu Li, Xing-Lou Yang, Aleksei A. Chmura, Guangjian Zhu, Jonathan H. Epstein, Jonna K. Mazet et al. “Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.” Nature 503, no. 7477 (2013): 535-538.

18. Yang, Yang, Chang Liu, Lanying Du, Shibo Jiang, Zhengli Shi, Ralph S. Baric, and Fang Li. “Two mutations were critical for bat-to-human transmission of Middle East respiratory syndrome coronavirus.” Journal of virology 89, no. 17 (2015): 9119-9123.

19. Menachery, Vineet D., Boyd L. Yount Jr, Kari Debbink, Sudhakar Agnihothram, Lisa E. Gralinski, Jessica A. Plante, Rachel L. Graham et al. “A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence.” Nature medicine 21, no. 12 (2015): 1508.

20. Menachery, V.D., Yount, B.L Jr, Debbink, K., Agnihothram, S., Gralinski, L.E., Plante, J.A., Graham, R.L., Scobey, T., Ge, X-Y., Donaldson, E.F., Randell, S.H., Lanzavecchia, A., Marasco, W.A., Shi, Z-L., & Baric, R.S. (2015). A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nature Medicine, 21, 1508– 1513. Doi: 10.1038/nm.3985 – Funding for this US/China joint chimeric research was supported by grants from the National Institute of Allergy & Infectious Disease, the National Institute of Aging of the US National Institutes of Health (NIH), the National Natural Science Foundation of China, and by USAID-EPT-PREDICT funding from EcoHealth Alliance.

Researchers and their affiliated institutions:

∙ Vineet D Menachery, Department of Epidemiology, University of North Carolina at Chapel Hill, USA.

∙ Boyd L Yount Jr, Department of Epidemiology, University of North Carolina at Chapel Hill, USA.

∙ Kari Debbink, Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, USA.

∙ Lisa E Gralinski, Department of Epidemiology, University of North Carolina at Chapel Hill, USA.

∙ Jessica A Plante, Department of Epidemiology, University of North Carolina at Chapel Hill, USA.

∙ Rachel L Graham, Department of Epidemiology, University of North Carolina at Chapel Hill, USA.

∙ Trevor Scobey, Department of Epidemiology, University of North Carolina at Chapel Hill, USA.

∙ Xing-Yi Ge, Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

∙ Eric F Donaldson, Department of Epidemiology, University of North Carolina at Chapel Hill, USA.

∙ Scott H Randell, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, USA.

∙ Antonio Lanzavecchia, Institute for Research in Biomedicine, Bellinzona Institute of Microbiology, Zurich, Switzerland.

∙ Wayne A Marasco, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA

∙ Zhengli-Li Shi, Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

∙ Ralph S Baric, Department of Epidemiology, University of North Carolina at Chapel Hill, USA

21. IBID. Menachery, V.D.

22. Cowling, Benjamin J., and Hiroshi Nishiura. “Virus interference and estimates of influenza vaccine effectiveness from test-negative studies.” Epidemiology 23, no. 6 (2012): 930-931.

23. Viboud, Cecile, and Lone Simonsen. “Does seasonal influenza vaccination increase the risk of illness with the 2009 A/H1N1 pandemic virus?.” Plos medicine 7, no. 4 (2010).

24. Janjua, Naveed Z., Danuta M. Skowronski, Travis S. Hottes, William Osei, Evan Adams, Martin Petric, Suzana Sabaiduc et al. “Seasonal influenza vaccine and increased risk of pandemic A/H1N1-related illness: first detection of the association in British Columbia, Canada.” Clinical Infectious Diseases 51, no. 9 (2010): 1017-1027.

25. Rikin, Sharon, Haomiao Jia, Celibell Y. Vargas, Yaritza Castellanos de Belliard, Carrie Reed, Philip LaRussa, Elaine L. Larson, Lisa Saiman, and Melissa S. Stockwell. “Assessment of temporally-related acute respiratory illness following influenza vaccination.” Vaccine 36, no. 15 (2018): 1958-1964.

26. Skowronski, Danuta M., Gaston De Serres, Natasha S. Crowcroft, Naveed Z. Janjua, Nicole Boulianne, Travis S. Hottes, Laura C. Rosella et al. “Association between the 2008–09 seasonal influenza vaccine and pandemic H1N1 illness during spring–summer 2009: four observational studies from Canada.” PLoS medicine 7, no. 4 (2010).

27. Wolff, Greg G. “Influenza vaccination and respiratory virus interference among Department of Defense personnel during the 2017–2018 influenza season.” Vaccine 38, no. 2 (2020): 350-354.

28. Butler, Declan. “Engineered bat virus stirs debate over risky research.” Nature News.



31. Li, Xingguang, Junjie Zai, Qiang Zhao, Qing Nie, Yi Li, Brian T. Foley, and Antoine Chaillon. “Evolutionary history, potential intermediate animal host, and cross‐species analyses of SARS‐CoV‐2.” Journal of medical virology (2020).


33. Zhou, P., Yang, X., Wang, X. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020).

34. Andersen, Kristian G., Andrew Rambaut, W. Ian Lipkin, Edward C. Holmes, and Robert F. Garry. “The proximal origin of SARS-CoV-2.” Nature medicine 26, no. 4 (2020): 450-452.

35. IBID Anderson, K.

36. Wu, C., Yang, Y., Liu, Y., Zhang, P., Wang, Y., Wang, Q., Xu, Y., Li, M., Zheng, M., Chen, L., & Li, H. (2020). Furin, a potential therapeutic target for COVID-19. Retrieved 14th May 2020 from

37. Ortega, Joseph Thomas, Maria Luisa Serrano, Flor Helene Pujol, and Hector Rafael Rangel. “Role of changes in SARS-CoV-2 spike protein in the interaction with the human ACE2 receptor: An in silico analysis.” EXCLI journal 19 (2020): 410.

38. Ren, W., Qu, X., Li, W., Han, Z., Yu, M., Zhou, P., Zhang, S-Y., Wang, L-F., Deng, H., & Shi, Z. (2008). Difference in Receptor Usage between Severe Acute Respiratory Syndrome (SARS) Coronavirus and SARS-Like Coronavirus of Bat Origin. Journal of Virology, 82 (4), 1899-1907: doi:10.1128/JVI.01085-07.




42. Jackson, R.J. et al., 2001. Expression of mouse interleukin-4 by a recombinant ectromelia virus overcomes genetic resistance to mousepox. Journal of Virology, 75, pp. 1205-1210.

43. Cello, J. et al., 2002. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science, 297, pp. 1016-1018.

44. Tumpey, T.M. et al. 2005. Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science, 310(5745), pp. 77–80.