2005 Study HIH-Chloroquine is a Potent Inhibitor of SARS Coronavirus Infection and Spread
Another lie and cover-up from Dr. Tony Fauci. This research published in 2005 was in The Virology Journal, the NIH’s prime document to post virology studies.
Dr. Fauci is a useful idiot to left and MSM. Here’s what is known about his liberal ties and wrong analysis. The two “experts” advising the President have finite experience in day-to-day management of patients with medical conditions. They practiced medicine for a combined five years prior to devoting their careers to research (Fauci-2 years). Fauci has never treated a patient with a virus like Covid-19. Why are pandemic recommendations coming from the sterile universe of virology research and not medical professionals dealing with real people?
The National Institute of Allergy and Infectious Diseases (NIAID), under Director Dr. Fauci, funded research at the Wuhan Institute of Virology. Why was NIAID allowed to earmark $3.7 million of taxpayer money to conduct risky research at the Chinese government's bioweapons laboratory?
In 2010, the Gates Foundation, which has cozy relationships with Wuhan University and the WHO, established the Decade of Vaccines Collaboration Program. The program’s Leadership Council includes Dr. Fauci, Director of NIAID. The Gates Foundation provided a grant of $499,944 to Wuhan University in 2018. Will Dr. Fauci work jointly with the Gates Foundation and Chinese to sell us the vaccine for the disease that originated in China? Follow the money.
Hydroxychloroquine (HCQ) is cheap, safe, plentiful and successful in curing COVID-19 when taken early in the disease process, according to many anecdotal reports. It’s safe use has spanned 65 years. Why is the federal government discouraging hydroxychloroquine’s use and promoting expensive Remdesivir? Uganda has population of 45 million and have used HCQ for treating COVID-19 in some hospitals. They have three deaths from the virus.
HCQ is cheap, meaning you can obtain with a prescription and private insurance. The cost for hydroxychloroquine oral tablet 200 mg is around $37 for a supply of 100 tablets.
Gilead Sciences Statement on NEJM Publication of Remdesivir Data from NIAID Study that Dr. Fauci supports. Foster City, Calif., May 22, 2020 – Gilead Sciences today issued the following statement from Merdad Parsey, MD, PhD, Chief Medical Officer, Gilead Sciences, on data from the National Institute of Allergy and Infectious Diseases’ (NIAID) study of our investigational antiviral drug Remdesivir, published in The New England Journal of Medicine (NEJM). Gilead announced June 29th much-anticipated pricing for its coronavirus treatment Remdesivir, saying it will cost hospitals $3,120 for a typical U.S. patient with commercial insurance. Follow the money ($37 or $3,120). No wonder Fauci is so cozy with Gilead.
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Chloroquine is a potent inhibitor of SARS coronavirus infection and spread
- Martin J Vincent,
- Eric Bergeron,
- Suzanne Benjannet,
- Bobbie R Erickson,
- Pierre E Rollin,
- Thomas G Ksiazek,
- Nabil G Seidah &
- Stuart T Nichol
Virology Journal volume 2, Article number: 69 (2005) Cite this article
Abstract
Background
Severe acute respiratory syndrome (SARS) is caused by a newly discovered coronavirus (SARS-CoV). No effective prophylactic or post-exposure therapy is currently available.
Results
We report, however, that chloroquine has strong antiviral effects on SARS-CoV infection of primate cells. These inhibitory effects are observed when the cells are treated with the drug either before or after exposure to the virus, suggesting both prophylactic and therapeutic advantage. In addition to the well-known functions of chloroquine such as elevations of endosomal pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, angiotensin-converting enzyme 2. This may negatively influence the virus-receptor binding and abrogate the infection, with further ramifications by the elevation of vesicular pH, resulting in the inhibition of infection and spread of SARS CoV at clinically admissible concentrations.
Conclusion
Chloroquine is effective in preventing the spread of SARS CoV in cell culture. Favorable inhibition of virus spread was observed when the cells were either treated with chloroquine prior to or after SARS CoV infection. In addition, the indirect immunofluorescence assay described herein represents a simple and rapid method for screening SARS-CoV antiviral compounds.
Background
Severe acute respiratory syndrome (SARS) is an emerging disease that was first reported in Guangdong Province, China, in late 2002. The disease rapidly spread to at least 30 countries within months of its first appearance, and concerted worldwide efforts led to the identification of the etiological agent as SARS coronavirus (SARS-CoV), a novel member of the family Coronaviridae [1]. Complete genome sequencing of SARS-CoV [2, 3] confirmed that this pathogen is not closely related to any of the previously established coronavirus groups. Budding of the SARS-CoV occurs in the Golgi apparatus [4] and results in the incorporation of the envelope spike glycoprotein into the virion. The spike glycoprotein is a type I membrane protein that facilitates viral attachment to the cellular receptor and initiation of infection, and angiotensin-converting enzyme-2 (ACE2) has been identified as a functional cellular receptor of SARS-CoV [5]. We have recently shown that the processing of the spike protein was effected by furin-like convertases and that inhibition of this cleavage by a specific inhibitor abrogated cytopathicity and significantly reduced the virus titer of SARS-CoV [6].
Due to the severity of SARS-CoV infection, the potential for rapid spread of the disease, and the absence of proven effective and safe in vivo inhibitors of the virus, it is important to identify drugs that can effectively be used to treat or prevent potential SARS-CoV infections. Many novel therapeutic approaches have been evaluated in laboratory studies of SARS-CoV: notable among these approaches are those using siRNA [7], passive antibody transfer [8], DNA vaccination [9], vaccinia or parainfluenza virus expressing the spike protein [10, 11], interferons [12, 13], and monoclonal antibody to the S1-subunit of the spike glycoprotein that blocks receptor binding [14]. In this report, we describe the identification of chloroquine as an effective pre- and post-infection antiviral agent for SARS-CoV. Chloroquine, a 9-aminoquinoline that was identified in 1934, is a weak base that increases the pH of acidic vesicles. When added extracellularly, the non-protonated portion of chloroquine enters the cell, where it becomes protonated and concentrated in acidic, low-pH organelles, such as endosomes, Golgi vesicles, and lysosomes. Chloroquine can affect virus infection in many ways, and the antiviral effect depends in part on the extent to which the virus utilizes endosomes for entry. Chloroquine has been widely used to treat human diseases, such as malaria, amoebiosis, HIV, and autoimmune diseases, without significant detrimental side effects [15]. Together with data presented here, showing virus inhibition in cell culture by chloroquine doses compatible with patient treatment, these features suggest that further evaluation of chloroquine in animal models of SARS-CoV infection would be warranted as we progress toward finding effective antivirals for prevention or treatment of the disease.
Results
Preinfection chloroquine treatment renders Vero E6 cells refractory to SARS-CoV infection
In order to investigate if chloroquine might prevent SARS-CoV infection, permissive Vero E6 cells [1] were pretreated with various concentrations of chloroquine (0.1–10 μM) for 20–24 h prior to virus infection. Cells were then infected with SARS-CoV, and virus antigens were visualized by indirect immunofluorescence as described in Materials and Methods. Microscopic examination (Fig. 1A) of the control cells (untreated, infected) revealed extensive SARS-CoV-specific immunostaining of the monolayer. A dose-dependant decrease in virus antigen-positive cells was observed starting at 0.1 μM chloroquine, and concentrations of 10 μM completely abolished SARS-CoV infection. For quantitative purposes, we counted the number of cells stained positive from three random locations on a slide. The average number of positively stained control cells was scored as 100% and was compared with the number of positive cells observed under various chloroquine concentrations (Fig. 1B). Pretreatment with 0.1, 1, and 10 μM chloroquine reduced infectivity by 28%, 53%, and 100%, respectively. Reproducible results were obtained from three independent experiments. These data demonstrated that pretreatment of Vero E6 cells with chloroquine rendered these cells refractory to SARS-CoV infection.
Figure 1
Prophylactic effect of chloroquine. Vero E6 cells pre-treated with chloroquine for 20 hrs. Chloroquine-containing media were removed and the cells were washed with phosphate buffered saline before they were infected with SARS-CoV (0.5 multiplicity of infection) for 1 h. in the absence of chloroquine. Virus was then removed and the cells were maintained in Opti-MEM (Invitrogen) for 16–18 h in the absence of chloroquine. SARS-CoV antigens were stained with virus-specific HMAF, followed by FITC-conjugated secondary antibodies. (A) The concentration of chloroquine used is indicated on the top of each panel. (B) SARS-CoV antigen-positive cells at three random locations were captured by using a digital camera, the number of antigen-positive cells was determined, and the average inhibition was calculated. Percent inhibition was obtained by considering the untreated control as 0% inhibition. The vertical bars represent the range of SEM.
Postinfection chloroquine treatment is effective in preventing the spread of SARS-CoV infection
In order to investigate the antiviral properties of chloroquine on SARS-CoV after the initiation of infection, Vero E6 cells were infected with the virus and fresh medium supplemented with various concentrations of chloroquine was added immediately after virus adsorption. Infected cells were incubated for an additional 16–18 h, after which the presence of virus antigens was analyzed by indirect immunofluorescence analysis. When chloroquine was added after the initiation of infection, there was a dramatic dose-dependant decrease in the number of virus antigen-positive cells (Fig. 2A). As little as 0.1–1 μM chloroquine reduced the infection by 50% and up to 90–94% inhibition was observed with 33–100 μM concentrations (Fig. 2B). At concentrations of chloroquine in excess of 1 μM, only a small number of individual cells were initially infected, and the spread of the infection to adjacent cells was all but eliminated. A half-maximal inhibitory effect was estimated to occur at 4.4 ± 1.0 μM chloroquine (Fig. 2C). These data clearly show that addition of chloroquine can effectively reduce the establishment of infection and spread of SARS-CoV if the drug is added immediately following virus adsorption.
Figure 2
Post-infection chloroquine treatment reduces SARS-CoV infection and spread. Vero E6 cells were seeded and infected as described for Fig. 1 except that chloroquine was added only after virus adsorption. Cells were maintained in Opti-MEM (Invitrogen) containing chloroquine for 16–18 h, after which they were processed for immunofluorescence. (A) The concentration of chloroquine is indicated on the top. (B) Percent inhibition and SEM were calculated as in Fig. 1B. (C) The effective dose (ED50) was calculated using commercially available software (Grafit, version 4, Erithacus Software).
Electron microscopic analysis indicated the appearance of significant amounts of extracellular virus particles 5–6 h after infection [16]. Since we observed antiviral effects by chloroquine immediately after virus adsorption, we further extended the analysis by adding chloroquine 3 and 5 h after virus adsorption and examined for the presence of virus antigens after 20 h. We found that chloroquine was still significantly effective even when added 5 h after infection (Fig. 3); however, to obtain equivalent antiviral effect, a higher concentration of chloroquine was required if the drug was added 3 or 5 h after adsorption.
Figure 3
Timed post-infection treatment with chloroquine. This experiment is similar to that depicted in Fig. 2 except that cells were infected at 1 multiplicity of infection, and chloroquine (10, 25, and 50 μM) was added 3 or 5 h after infection.
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