Let me get this straight about the COVID-19 vaccine

Let me get this straight about the COVID-19 vaccine


Updates:The C-19 shot is a global time bomb.

​

​

The first-ever autopsy of a person vaccinated against COVID-19, has revealed viral RNA in almost every organ of the body. The vaccine, while triggering an immune response, DID NOT STOP the virus from entering every organ in the body.​

The viral RNA was found in virtually every organ in the body, which means the spike proteins as well.
There are antibodies (like the “vaccine” is supposed to create) but they’re irrelevant because, based on a study from Japan, we now know that the spike S1 protein is what does the damage.
We spoke to one Infectious Disease specialist from a hospital in New Jersey this morning. We sent the actual autopsy findings to him and asked for his thoughts.
When he called back a while later, he was clearly shaken-up. He told us “You cannot quote me by name, I will get fired by the hospital if you do.” We agreed to conceal his identity.
He then told us:
“People think that only a MINORITY of people get adverse effects from the vaccine.
Based on this new research, it means that everyone – EVENTUALLY -will have adverse effects, because those spike proteins will be binding to ACE2 receptors everywhere in the body.
That mRNA was supposed to stay in the injection site and it’s not. That means the spike proteins created by the mRNA will be in every organ as well, and we now know it is the spike proteins that do the damage.
Worse, the viral RNA being found in every organ despite a vaccine, indicates either:
1) The vaccine doesn’t work at all, OR;
2) The virus is enjoying Antibody Dependent Enhancement (ADE), meaning it actually spreads FASTER in vaccinated people.
This is a GLOBAL TIMEBOMB.”
According to the published postmortem report, the vaccinated man was 86 years old and tested NEGATIVE for COVID-19 when first admitted to the hospital with severe gastro-intestinal trouble and difficulty breathing. Here is what the reports describes:
We report on an 86-year-old male resident of a retirement home who received vaccine against SARS-CoV-2. Past medical history included systemic arterial hypertension, chronic venous insufficiency, dementia and prostate carcinoma. On January 9, 2021, the man received lipid nanoparticle-formulated, nucleoside-modified RNA vaccine BNT162b2 in a 30 μg dose. On that day and in the following 2 weeks, he presented with no clinical symptoms (Table 1). On day 18, he was admitted to hospital for worsening diarrhea. Since he did not present with any clinical signs of COVID-19, isolation in a specific setting did not occur. Laboratory testing revealed hypochromic anemia and increased creatinine serum levels. Antigen test and polymerase chain reaction (PCR) for SARS-CoV-2 were negative.
The report of the postmortem makes clear tests showed “no morphological changes associated with COVID” in his organs.
“Morphological” means structural. COVID infection is now known to cause very specific structural changes to the places it infects. THOSE CHANGES HAD NOT APPEARED in the vaccinated man before he died.
The now dead vaccinated man was in a room where another patient ultimately tested POSTIVE for COVID, and the report states they think the dead vaccinated man caught COVID after he was admitted, from the other patient in the same room.
So the damage to the organs of the now dead vaccine recipient, took place BEFORE he was infected with COVID by the other hospital room patient.
Worse, once the vaccinated man actually got COVID, it spread so fast within his body, he apparently never stood a chance. Here are tissue images:
postmortem-tissue-images-1.jpg

The full postmortem report is published at ScienceDirect.com (HERE)
HAL TURNER COMMENTARY
I am not a Doctor or a Scientist so I cannot offer a competent medical analysis, but as a layman, from where I sit, this doesn’t look so good.
The Postmortem report says clearly “ These results indicate that the patient had already developed relevant immunogenicity through vaccination” yet he got infected by another patient at day 24 (after vax) upon being admitted to the hospital, and died 4 days later.
If one reads the entire article, the whole story is: Patient was given the vaccine, it got him hospitalized with ulcerative colitis due to blood clots, during his hospitalization he got infected by an asymptomatic hospital room mate, and died 4 days later.
My conclusion: the jab drove this elderly man to his end.
I might be wrong, but this is what I see from the postmortem report.
I have not taken the vax and I do not intend to take it.
Throughout this entire COVID debacle, the one constant is that the disease has a 99.8% SURVIVAL rate. To me, it’s not a pandemic. To me it’s not even worth getting a vaccine; I think my immune system will handle it just fine.
Given all the adverse reactions to the vax, I now firmly conclude that getting the vax is a far more dangerous thing than getting the actual illness.
God gave me an immune system. I think I will trust God on this. After all, I truly believe I will not live one micro-second longer than God wants, and conversely, I will not die one micro-second sooner than God wants.
Faith is not “belief without proof” it is “trust without reservation.” I trust God.
SUDDEN DEATH of 32 YEAR OLD
This couple’s step-son, age 32, died from a heart attack 12 hours after getting the Johnson & Johnson Vaccine. Coroner refuses to release reports; calls cause of death “inconclusive.” A lot of these deaths are simply being covered up. Here’s the family to tell you:


___
"Global Time Bomb" First case of postmortem study of patient vaccinated against SARS-CoV-2; "viral RNA found in every organ of the body"
 
IMG_20210703_223529_801.jpg
It doesn't need a rocket scientist to know that there is a secret agenda behind this vaccine sensitization. Only a lunatic would take part of this experiment which would not only alter our genome for subsequent generations but also is an abomination to our creator.
 
Mathanzua said:

Updates:The C-19 shot is a global time bomb.

​

​

The first-ever autopsy of a person vaccinated against COVID-19, has revealed viral RNA in almost every organ of the body. The vaccine, while triggering an immune response, DID NOT STOP the virus from entering every organ in the body.​

The viral RNA was found in virtually every organ in the body, which means the spike proteins as well.
There are antibodies (like the “vaccine” is supposed to create) but they’re irrelevant because, based on a study from Japan, we now know that the spike S1 protein is what does the damage.
We spoke to one Infectious Disease specialist from a hospital in New Jersey this morning. We sent the actual autopsy findings to him and asked for his thoughts.
When he called back a while later, he was clearly shaken-up. He told us “You cannot quote me by name, I will get fired by the hospital if you do.” We agreed to conceal his identity.
He then told us:
“People think that only a MINORITY of people get adverse effects from the vaccine.
Based on this new research, it means that everyone – EVENTUALLY -will have adverse effects, because those spike proteins will be binding to ACE2 receptors everywhere in the body.
That mRNA was supposed to stay in the injection site and it’s not. That means the spike proteins created by the mRNA will be in every organ as well, and we now know it is the spike proteins that do the damage.
Worse, the viral RNA being found in every organ despite a vaccine, indicates either:
1) The vaccine doesn’t work at all, OR;
2) The virus is enjoying Antibody Dependent Enhancement (ADE), meaning it actually spreads FASTER in vaccinated people.
This is a GLOBAL TIMEBOMB.”
According to the published postmortem report, the vaccinated man was 86 years old and tested NEGATIVE for COVID-19 when first admitted to the hospital with severe gastro-intestinal trouble and difficulty breathing. Here is what the reports describes:
We report on an 86-year-old male resident of a retirement home who received vaccine against SARS-CoV-2. Past medical history included systemic arterial hypertension, chronic venous insufficiency, dementia and prostate carcinoma. On January 9, 2021, the man received lipid nanoparticle-formulated, nucleoside-modified RNA vaccine BNT162b2 in a 30 μg dose. On that day and in the following 2 weeks, he presented with no clinical symptoms (Table 1). On day 18, he was admitted to hospital for worsening diarrhea. Since he did not present with any clinical signs of COVID-19, isolation in a specific setting did not occur. Laboratory testing revealed hypochromic anemia and increased creatinine serum levels. Antigen test and polymerase chain reaction (PCR) for SARS-CoV-2 were negative.
The report of the postmortem makes clear tests showed “no morphological changes associated with COVID” in his organs.
“Morphological” means structural. COVID infection is now known to cause very specific structural changes to the places it infects. THOSE CHANGES HAD NOT APPEARED in the vaccinated man before he died.
The now dead vaccinated man was in a room where another patient ultimately tested POSTIVE for COVID, and the report states they think the dead vaccinated man caught COVID after he was admitted, from the other patient in the same room.
So the damage to the organs of the now dead vaccine recipient, took place BEFORE he was infected with COVID by the other hospital room patient.
Worse, once the vaccinated man actually got COVID, it spread so fast within his body, he apparently never stood a chance. Here are tissue images:
postmortem-tissue-images-1.jpg

The full postmortem report is published at ScienceDirect.com (HERE)
HAL TURNER COMMENTARY
I am not a Doctor or a Scientist so I cannot offer a competent medical analysis, but as a layman, from where I sit, this doesn’t look so good.
The Postmortem report says clearly “ These results indicate that the patient had already developed relevant immunogenicity through vaccination” yet he got infected by another patient at day 24 (after vax) upon being admitted to the hospital, and died 4 days later.
If one reads the entire article, the whole story is: Patient was given the vaccine, it got him hospitalized with ulcerative colitis due to blood clots, during his hospitalization he got infected by an asymptomatic hospital room mate, and died 4 days later.
My conclusion: the jab drove this elderly man to his end.
I might be wrong, but this is what I see from the postmortem report.
I have not taken the vax and I do not intend to take it.
Throughout this entire COVID debacle, the one constant is that the disease has a 99.8% SURVIVAL rate. To me, it’s not a pandemic. To me it’s not even worth getting a vaccine; I think my immune system will handle it just fine.
Given all the adverse reactions to the vax, I now firmly conclude that getting the vax is a far more dangerous thing than getting the actual illness.
God gave me an immune system. I think I will trust God on this. After all, I truly believe I will not live one micro-second longer than God wants, and conversely, I will not die one micro-second sooner than God wants.
Faith is not “belief without proof” it is “trust without reservation.” I trust God.
SUDDEN DEATH of 32 YEAR OLD
This couple’s step-son, age 32, died from a heart attack 12 hours after getting the Johnson & Johnson Vaccine. Coroner refuses to release reports; calls cause of death “inconclusive.” A lot of these deaths are simply being covered up. Here’s the family to tell you:


___
"Global Time Bomb" First case of postmortem study of patient vaccinated against SARS-CoV-2; "viral RNA found in every organ of the body"
Click to expand...
You have moved me to search more information about vaccines.

There are several different types of vaccines. Each type is designed to teach our immune system how to fight off certain kinds of germs—and the serious diseases they cause.

When scientists create vaccines, they consider:
[emoji830]︎ How your immune system responds to the germ;
[emoji830]︎ Who needs to be vaccinated against the germ; and
[emoji830]︎ The best technology or approach to create the vaccine
Based on a number of these factors, scientists decide which type of vaccine they will make. There are several types of vaccines, including:
Inactivated vaccines;
Live-attenuated vaccines;
Messenger RNA (mRNA) vaccines;
Subunit, recombinant, polysaccharide and conjugate vaccines;
Toxoid vaccines; and
Viral vector vaccines;
Click to expand...

Inactivated vaccines use the killed version of the germ that causes a disease. Because these vaccines are so similar to the natural infection that they help prevent, they create a strong and long-lasting immune response. Just 1 or 2 doses of most live vaccines can give you a lifetime of protection against a germ and the disease it causes.

But live-attenuated vaccines also have some limitations. For example, because they contain a small amount of the weakened live virus, some people should talk to their health care provider before receiving them, such as people with weakened immune systems, long-term health problems, or people who’ve had an organ transplant. They need to be kept cool, so they don’t travel well. That means they can’t be used in countries with limited access to refrigerators. Live-attenuated vaccines are used to protect against: Measles, mumps, rubella (MMR combined vaccine); Rotavirus; Smallpox; Chickenpox; Yellow fever

With respect to Messenger RNA vaccines — also called mRNA vaccines, the subject of your post, researchers have been studying and working with them for decades and this technology was used to make some of the COVID-19 vaccines. mRNA vaccines make proteins in order to trigger an immune response. Although mRNA vaccines have several benefits compared to other types of vaccines, including shorter manufacturing times and, because they do not contain a live virus, the risk of causing disease in the person getting vaccinated have yet to be established.
 
Mathanzua said:
I am sorry kwa kukushambulia, lakini inauma kuona kwamba we have Africans like you,despite what the whites have done to us.There is something seriously wrong with your psychic Pythagoras.Kwa jinsi wazungu walivyotufanyia haingii akilini kwamba mtu,na hasa Mwafrika anaweza kuwapenda Wazungu.Sana sana nilitegemea kwamba having a President like Magufuli would give us a glime of hope not only as Tanzanians,but also as Africans .Badala yake unamshambulia,ajabu sana. Sina wasiwasi wote wote kwamba you are part of their "State Capture System."
Click to expand...
Huyo Yuko beyond salvaging Mkuu,Wenye macho tumekuelewa.
 
Updates:
Hili ndilo jabali lililojisombea sifa duniani kote kwa msimamo wake usioyumba kuhusu C-19.Mwishowe NWO wakaamua kumuua.Tangulia Magufuli na sisi tunakuja,hakuna aishiye milele.
 

Attachments

  • VID-20210711-WA0001.mp4
    2.1 MB
  • VID-20210711-WA0002.mp4
    2.3 MB

Updates:​

"You have never seen anything like this." A Surgeon who operated on young Italian vaccine victim reports.​



camilla.jpg

AstraZeneca victim Camilla Canepa.

The surgeon who operated an Italian girl, Camilla Canepa, who died at the age of 18 from the effects of the vaccine, said "he has never seen anything like this.It’s not normal,” he added.
Free West Media
Camilla Canepa was operated on by Gianluigi Zona, director of the neurosurgical and neuro-traumatological clinic of the San Martino hospital: “I had never seen a brain that was affected by such an extensive and severe thrombosis.”
The neurosurgeon on duty in San Martino that night was Alessandro d’Andrea, who also called the chief physician to his side at the operating table. “We decided to have a decompression craniotomy, in which the skull is opened to relieve internal pressure.”
Zona recounted the experience: “All venous sinuses were blocked with thrombi, a scenario I have never seen in my many years in this profession. Think of the venous sinus as the river in the middle of a valley where several streams converge. If a dam is built in the middle of the watercourse, the river swells and the tributaries can no longer drain at this point, so that the pressure rises upstream.
“I’m neither a virologist, nor an epidemiologist, nor a coroner, but given the image I saw in the girl’s head, it is clear that we are dealing with something that is not normal.”
The parents of the 18-year-old who died, told the media: “She had no disease.” Last week, the prosecutor of Genoa, which coordinates the ongoing investigations, will instruct the Pavia coroner to perform the autopsy on the body of Camilla Canepa, the eighteen year old who died after the vaccine. The girl allegedly suffered from chronic platelet deficiency, a “familial autoimmune thrombocytopenia”. This is what investigators have learned from the first reports of the doctors.
But the girl’s family, assisted by the lawyer Angelo Paone, is firm on this point. “Camilla had no disease,” explained their lawyer. Supporting their claim, are two different CT scans. She had undergone the two scans, and was discharged after the first one that had not shown the situation of the thrombosis in progress, but immediately transferred to the hospital after the second showing that her health had been compromised.
The girl arrived in the emergency room in the Lavagna hospital on June 3, just a week after theAstraZeneca shot. She had complained of severe headaches.
Canepa, who was from the town of Sestri Levante in Liguria, was given the jab during a vaccination “open day” for youths over 18. TheAstraZeneca jab has been approved for all over-18s, but in Italy it is only recommended for over-60s due to links to several cases of blood clots in younger people.
Camilla’s case has raised concerns among young people previously keen to get vaccinated to obtain the Green Pass or vaccine passport enabling them to travel and attend mass events.
___
Surgeon who operated on young Italian vaccine victim: ‘You have never seen anything like this’ | Free West Media
 
Updates:
A 2005 scientific report has confirmed that Chloroquine is a potent inhibitor of SARS coronavirus infection and spread,so there is absolutely no justification for the so called C-19 vaccine.So the aim is obviously to maim, debilitate, cause sickness and exterminate humanity.

The report was submitted to the Journal of Virology Journal volume 2, Article number: 69(2005) by the following authors:-
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 familyCoronaviridae [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 safein 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
figure1
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.
Full size image

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
figure2
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).
Full size image
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
figure3
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.
Full size image

Ammonium chloride inhibits SARS-CoV infection of Vero E6 cells​

Since chloroquine inhibited SARS-CoV infection when added before or after infection, we hypothesized that another common lysosomotropic agent, NH4Cl, might also function in a similar manner. Ammonium chloride has been widely used in studies addressing endosome-mediated virus entry. Coincidently, NH4Cl was recently shown to reduce the transduction of pseudotype viruses decorated with SARS-CoV spike protein [17, 18]. In an attempt to examine if NH4Cl functions similarly to chloroquine, we performed infection analyses in Vero E6 cells before (Fig. 4A) and after (Fig. 4B) they were treated with various concentrations of NH4Cl. In both cases, we observed a 93–99% inhibition with NH4Cl at ≥ 5 mM. These data indicated that NH4Cl (≥ 5 mM) and chloroquine (≥ 10 μM) are very effective in reducing SARS-CoV infection. These results suggest that effects of chloroquine and NH4Cl in controlling SARS CoV infection and spread might be mediated by similar mechanism(s).
Figure 4
figure4
NH 4 Cl inhibits SARS-CoV during pre or post infection treatment. NH4Cl was added to the cells either before (A) or after (B) infection, similar to what was done for chloroquine in Figs 1 and 2. Antigen-positive cells were counted, and the results were presented as in Fig. 1B.
Full size image

Effect of chloroquine and NH4Cl on cell surface expression of ACE2​

We performed additional experiments to elucidate the mechanism of SARS-CoV inhibition by chloroquine and NH4Cl. Since intra-vesicular acidic pH regulates cellular functions, including N-glycosylation trimming, cellular trafficking, and various enzymatic activities, it was of interest to characterize the effect of both drugs on the processing, glycosylation, and cellular sorting of SARS-CoV spike glycoprotein and its receptor, ACE2. Flow cytometry analysis was performed on Vero E6 cells that were either untreated or treated with highly effective anti-SARS-CoV concentrations of chloroquine or NH4Cl. The results revealed that neither drug caused a significant change in the levels of cell-surface ACE2, indicating that the observed inhibitory effects on SARS-CoV infection are not due to the lack of available cell-surface ACE2 (Fig.5A). We next analyzed the molecular forms of endogenous ACE2 in untreated Vero E6 cells and in cells that were pre-incubated for 1 h with various concentrations of either NH4Cl (2.5–10 mM) or chloroquine (1 and 10 μM) and labeled with 35S-(Met) for 3 h in the presence or absence of the drugs (Fig. 5B and5C). Under normal conditions, we observed two immunoreactive ACE2 forms, migrating at ~105 and ~113 kDa, respectively (Fig.5B, lane 1). The ~105-kDa protein is endoglycosidase H sensitive, suggesting that it represents the endoplasmic reticulum (ER) localized form, whereas the ~113-kDa protein is endoglycosidase H resistant and represents the Golgi-modified form of ACE2 [19]. The specificity of the antibody was confirmed by displacing the immunoreactive protein bands with excess cold-soluble human recombinant ACE2 (+ rhACE2; Fig. 5B, lane 2). When we analyzed ACE2 forms in the presence of NH4Cl, a clear stepwise increase in the migration of the ~113-kDa protein was observed with increasing concentrations of NH4Cl, with a maximal effect observed at 10 mM NH4Cl, resulting in only the ER form of ACE2 being visible on the gel (Fig. 5B, compare lanes 3–5). This suggested that the trimming and/or terminal modifications of the N-glycosylated chains of ACE2 were affected by NH4Cl treatment. In addition, at 10 mM NH4Cl, the ER form of ACE2 migrated with slightly faster mobility, indicating that NH4Cl at that concentration might also affect core glycosylation. We also examined the terminal glycosylation status of ACE2 when the cells were treated with chloroquine (Fig.5C). Similar to NH4Cl, a stepwise increase in the electrophoretic mobility of ACE2 was observed with increasing concentrations of chloroquine. At 25 μM chloroquine, the faster electrophoretic mobility of the Golgi-modified form of ACE2 was clearly evident. On the basis of the flow cytometry and immunoprecipitation analyses, it can be inferred that NH4Cl and chloroquine both impaired the terminal glycosylation of ACE2, while NH4Cl resulted in a more dramatic effect. Although ACE2 is expressed in similar quantities at the cell surface, the variations in its glycosylation status might render the ACE2-SARS-CoV interaction less efficient and inhibit virus entry when the cells are treated with NH4Cl and chloroquine.
Figure 5
figure5
Effect of lysomotropic agents on the cell-surface expression and biosynthesis of ACE2. (A) Vero E6 cells were cultured for 20 h in the absence (control) or presence of chloroquine (10 μM) or NH4Cl (20 mM). Cells were labeled with anti-ACE2 (grey histogram) or with a secondary antibody alone (white histogram). (B)Biosynthesis of ACE2 in untreated cells or in cells treated with NH4Cl. Vero E6 cells were pulse-labeled for 3 h with 35S-Met, and the cell lysates were immunoprecipitated with an ACE2 antibody (lane 1). Preincunbation of the antibody with recombinant human ACE2 (rhACE2) completely abolished the signal (lane 2). The positions of the endoglycosidase H-sensitive ER form and the endoglycosidase H-resistant Golgi form of ACE2 are emphasized. Note that the increasing concentration of NH4Cl resulting in the decrease of the Golgi form of ACE2. (C) A similar experiment was performed in the presence of the indicated concentrations of chloroquine. Note the loss of terminal glycans with increasing concentrations of chloroquine. (D)The terminal glycosidic modification of ACE2 was evaluated by neuraminidase treatment of immunoprecipitated ACE2. Here cells were treated with 1–25 μM concentrations of chloroquine during starvation, pulse, and 3-h chase.
Full size image
To confirm that ACE2 undergoes terminal sugar modifications and that the terminal glycosylation is affected by NH4Cl or chloroquine treatment, we performed immunopreipitation of 35S-labeled ACE2 and subjected the immunoprecipitates to neuraminidase digestion. Proteins were resolved using SDS-PAGE (Fig 5D). It is evident from the slightly faster mobility of the Golgi form of ACE2 after neuraminidase treatment (Fig 5D, compare lanes 1 and 2), that ACE2 undergoes terminal glycosylation; however, the ER form of ACE2 was not affected by neuraminidase. Cells treated with 10 μM chloroquine did not result in a significant shift; whereas 25 μM chloroquine caused the Golgi form of ACE2 to resolve similar to the neuraminidase-treated ACE2 (Fig5D, compare lanes 5 and 6). These data provide evidence that ACE2 undergoes terminal glycosylation and that chloroquine at anti-SARS-CoV concentrations abrogates the process.

Effect of chloroquine and NH4Cl on the biosynthesis and processing of SARS-CoV spike protein​

We next addressed whether the lysosomotropic drugs (NH4Cl and chloroquine) affect the biosynthesis, glycosylation, and/or trafficking of the SARS-CoV spike glycoprotein. For this purpose, Vero E6 cells were infected with SARS-CoV for 18 h. Chloroquine or ammonium chloride was added to these cells during while they were being starved (1 h), labeled (30 min) or chased (3 h). The cell lysates were analyzed by immunoprecipitation with the SARS-specific polyclonal antibody (HMAF). The 30-min pulse results indicated that pro-spike (proS) was synthesized as a ~190-kDa precursor (proS-ER) and processed into ~125-, ~105-, and ~80-kDa proteins (Fig. 6A, lane 2), a result identical to that in our previous analysis [6]. Except for the 100 μM chloroquine (Fig.6A, lane 3), there was no significant difference in the biosynthesis or processing of the virus spike protein in untreated or chloroquine-treated cells (Fig.6A, lanes 4–6). It should be noted that chloroquine at 100 μM resulted in an overall decrease in biosynthesis and in the levels of processed virus glycoprotein. In view of the lack of reduction in the biosynthesis and processing of the spike glycoprotein in the presence of chloroquine concentrations (10 and 50 μM) that caused large reductions in SARS-CoV replication and spread, we conclude that the antiviral effect is probably not due to alteration of virus glycoprotein biosynthesis and processing. Similar analyses were performed with NH4Cl, and the data suggested that the biosynthesis and processing of the spike protein were also not negatively affected by NH4Cl (Fig. 6A, lanes 7–12). Consistent with our previous analysis [6], we observed the presence of a larger protein, which is referred to here as oligomers. Recently, Song et al. [20] provided evidence that these are homotrimers of the SARS-CoV spike protein and were incorporated into the virions. Interestingly, the levels of the homotrimers in cells treated with 100 μM chloroquine and 40 and 20 mM NH4Cl (Fig. 6A, lanes 3, 9, and 10) were slightly lower than in control cells or cells treated with lower drug concentrations.
Figure 6
figure6
Effects of NH 4 Cl and chloroquine (CQ) on the biosynthesis, processing, and glycosylation of SARS-CoV spike protein. Vero E6 cells were infected with SARS-CoV as described in Fig. 2. CQ or NH4Cl was added during the periods of starvation (1 h) and labeling (30 min) with 35S-Cys and followed by chase for 3 h in the presence of unlabeled medium. Cells were lysed in RIPA buffer and immunoprecipitated with HMAF. Virus proteins were resolved using 3–8% NuPAGE gel (Invitrogen). The cells presented were labeled for 30 min (A) and chased for 3 h(B). The migration positions of the various spike molecular forms are indicated at the right side, and those of the molecular standards are shown to the left side. proS-ER and proS-Golgi are the pro-spike of SARS-Co in the ER and Golgi compartments, respectively and proS-ungly is the unglycosylated pro-spike ER.
Full size image
The data obtained from a 30-min pulse followed by a 3-h chase (Fig. 6B, lanes 2 and 8) confirmed our earlier observation that the SARS-CoV spike protein precursor (proS-ER) acquires Golgi-specific modifications (proS-Golgi) resulting in a ~210-kDa protein [6]. Chloroquine at 10, 25, and 50 μM had no substantial negative impact on the appearance of the Golgi form (Fig.6B, compare lane 2 to lanes 4–6). Only at 100 μM chloroquine was a reduction in the level of the Golgi-modified pro-spike observed (lane 3). On the other hand, NH4Cl abrogated the appearance of Golgi-modified forms at ≥10 mM (compare lane 8 with 9–11) and had a milder effect at 1 mM (lane 12). These data clearly demonstrate that the biosynthesis and proteolytic processing of SARS-CoV spike protein are not affected at chloroquine (25 and 50 μM) and NH4Cl (1 mM) doses that cause virus inhibitory effects. In addition, with 40, 20, and 10 mM NH4Cl, there was an increased accumulation of proS-ER with a concomitant decrease in the amount of oligomers (Fig. 6B, lanes 9–11). When we examined the homotrimers, we found that chloroquine at 100 μM and NH4Cl at 40 and 20 mM resulted in slightly faster mobility of the trimers (Fig. 6B, lanes 3, 9, and 10), but lower drug doses, which did exhibit significant antiviral effects, did not result in appreciable differences. These data suggest that the newly synthesized intracellular spike protein may not be a major target for chloroquine and NH4Cl antiviral action. The faster mobility of the trimer at certain higher concentration of the drugs might be due the effect of these drugs on the terminal glycosylation of the trimers.

Discussion​

We have identified chloroquine as an effective antiviral agent for SARS-CoV in cell culture conditions, as evidenced by its inhibitory effect when the drug was added prior to infection or after the initiation and establishment of infection. The fact that chloroquine exerts an antiviral effect during pre- and post-infection conditions suggest that it is likely to have both prophylactic and therapeutic advantages. Recently, Keyaerts et al. [21] reported the antiviral properties of chloroquine and identified that the drug affects SARS-CoV replication in cell culture, as evidenced by quantitative RT-PCR. Taken together with the findings of Keyaerts et al. [21], our analysis provides further evidence that chloroquine is effective against SARS-CoV Frankfurt and Urbani strains. We have provided evidence that chloroquine is effective in preventing SARS-CoV infection in cell culture if the drug is added to the cells 24 h prior to infection. In addition, chloroquine was significantly effective even when the drug was added 3–5 h after infection, suggesting an antiviral effect even after the establishment of infection. Since similar results were obtained by NH4Cl treatment of Vero E6 cells, the underlying mechanism(s) of action of these drugs might be similar.
Apart from the probable role of chloroquine on SARS-CoV replication, the mechanisms of action of chloroquine on SARS-CoV are not fully understood. Previous studies have suggested the elevation of pH as a mechanism by which chloroquine reduces the transduction of SARS-CoV pseudotype viruses [17, 18]. We examined the effect of chloroquine and NH4Cl on the SARS-CoV spike proteins and on its receptor, ACE2. Immunoprecipitation results of ACE2 clearly demonstrated that effective anti-SARS-CoV concentrations of chloroquine and NH4Cl also impaired the terminal glycosylation of ACE2. However, the flow cytometry data demonstrated that there are no significant differences in the cell surface expression of ACE2 in cells treated with chloroquine or NH4Cl. On the basis of these results, it is reasonable to suggest that the pre-treatment with NH4Cl or chloroquine has possibly resulted in the surface expression of the under-glycosylated ACE2. In the case of chloroquine treatment prior to infection, the impairment of terminal glycosylation of ACE2 may result in reduced binding affinities between ACE2 and SARS-CoV spike protein and negatively influence the initiation of SARS-CoV infection. Since the biosynthesis, processing, Golgi modification, and oligomerization of the newly synthesized spike protein were not appreciably affected by anti-SARS-CoV concentrations of either chloroquine or NH4Cl, we conclude that these events occur in the cell independent of the presence of the drugs. The potential contribution of these drugs in the elevation of endosomal pH and its impact on subsequent virus entry or exit could not be ruled out. A decrease in SARS-CoV pseudotype transduction in the presence of NH4Cl was observed and was attributed to the effect on intracellular pH [17, 18]. When chloroquine or NH4Cl are added after infection, these agents can rapidly raise the pH and subvert on-going fusion events between virus and endosomes, thus inhibiting the infection.
In addition, the mechanism of action of NH4Cl and chloroquine might depend on when they were added to the cells. When added after the initiation of infection, these drugs might affect the endosome-mediated fusion, subsequent virus replication, or assembly and release. Previous studies of chloroquine have demonstrated that it has multiple effects on mammalian cells in addition to the elevation of endosomal pH, including the prevention of terminal glycosyaltion of immunoglobulins [22]. When added to virus-infected cells, chloroquine inhibited later stages in vesicular stomatitis virus maturation by inhibiting the glycoprotein expression at the cell surface [23], and it inhibited the production of infectious HIV-1 particles by interfering with terminal glycosylation of the glycoprotein [24,25]. On the basis of these properties, we suggest that the cell surface expression of under-glycosylated ACE2 and its poor affinity to SARS-CoV spike protein may be the primary mechanism by which infection is prevented by drug pretreatment of cells prior to infection. On the other hand, rapid elevation of endosomal pH and abrogation of virus-endosome fusion may be the primary mechanism by which virus infection is prevented under post-treatment conditions. More detailed SARS CoV spike-ACE2 binding assays in the presence or absence of chloroquine will be performed to confirm our findings. Our studies indicate that the impact of NH4Cl and chloroquine on the ACE2 and spike protein profiles are significantly different. NH4Cl exhibits a more pronounced effect than does chloroquine on terminal glycosylation, highlighting the novel intricate differences between chloroquine and ammonium chloride in affecting the protein transport or glycosylation of SARS-CoV spike protein and its receptor, ACE2, despite their well-established similar effects of endosomal pH elevation.
The infectivity of coronaviruses other than SARS-CoV are also affected by chloroquine, as exemplified by the human CoV-229E [15]. The inhibitory effects observed on SARS-CoV infectivity and cell spread occurred in the presence of 1–10 μM chloroquine, which are plasma concentrations achievable during the prophylaxis and treatment of malaria (varying from 1.6–12.5 μM) [26] and hence are well tolerated by patients. It recently was speculated that chloroquine might be effective against SARS and the authors suggested that this compound might block the production of TNFα, IL6, or IFNγ [15]. Our data provide evidence for the possibility of using the well-established drug chloroquine in the clinical management of SARS.

Conclusion​

Chloroquine, a relatively safe, effective and cheap drug used for treating many human diseases including malaria, amoebiosis and human immunodeficiency virus is effective in inhibiting the infection and spread of SARS CoV in cell culture. The fact that the drug has significant inhibitory antiviral effect when the susceptible cells were treated either prior to or after infection suggests a possible prophylactic and therapeutic use.

Methods​

SARS-CoV infection, immunofluorescence, and immunoprecipitation analyses​

Vero E6 cells (an African green monkey kidney cell line) were infected with SARS-CoV (Urbani strain) at a multiplicity of infection of 0.5 for 1 h. The cells were washed with PBS and then incubated in OPTI-MEM (Invitrogen) medium with or without various concentrations of either chloroquine or NH4Cl (both from Sigma). Immunofluorescence staining was performed with SARS-CoV-specific hyperimmune mouse ascitic fluid (HMAF) [8] followed by anti-mouse fluorescein-coupled antibody.
Eighteen hours after infection, the virus-containing supernatants were removed, and the cells were pulsed with 35S-(Cys) for 30 min and chased for 3 h before lysis in RIPA buffer. Clarified cell lysates and media were incubated with HMAF, and immunoprecipitated proteins were separated by 3–8% NuPAGE gel (Invitrogen); proteins were visualized by autoradiography. In some experiments, cells were chased for 3 h with isotope-free medium. Clarified cell supernatants were also immunoprecipitated with SARS-CoV-specific HMAF.

ACE2 flow cytometry analysis and biosynthesis​

Vero E6 cells were seeded in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum. The next day, the cells were incubated in Opti-MEM (Invitrogen) in the presence or absence of 10 μM chloroquine or 20 mM NH4Cl. To analyze the levels of ACE2 at the cell surface, cells were incubated on ice with 10 μg/mL affinity-purified goat anti-ACE2 antibody (R&D Systems) and then incubated with FITC-labeled swine anti-goat IgG antibody (Caltag Laboratories). Labeled cells were analyzed by flow cytometry with a FACSCalibur flow cytometer (BD Biosciences). For ACE2 biosynthesis studies, Vero E6 cells were pulsed with 250 μCi 35S-(Met) (Perkin Elmer) for 3 h with the indicated concentrations of chloroquine or NH4Cl and then lysed in RIPA buffer. Clarified lysates were immunoprecipitated with an affinity-purified goat anti-ACE2 antibody (R&D systems), and the immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis.

References​

  1. 1.
    Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota PB, Fields B, DeRisi J, Yang JY, Cox N, Hughes J, LeDuc JW, Bellini WJ, Anderson LJ, SARS Working Group: A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 2003, 348:1953-1966. 10.1056/NEJMoa030781
    CAS Article PubMed Google Scholar
  2. 2.
    Marra MA, Jones SJ, Astell CR, Holt RA, Brooks-Wilson A, Butterfield YS, Khattra J, Asano JK, Barber SA, Chan SY, Cloutier A, Coughlin SM, Freeman D, Girn N, Griffith OL, Leach SR, Mayo , McDonald H, Montgomery SB, Pandoh PK, Petrescu AS, Robertson AG, Schein JE, Siddiqui A, Smailus DE, Stott JM, Yang GS, Plummer F, Andonov A, Artsob H, Bastien N, Bernard K, Booth TF, Bowness D, Czub M, Drebot M, Fernando L, Flick R, Garbutt M, Gray M, Grolla A, Jones S, Feldmann H, Meyers A, Kabani A, Li Y, Normand S, Stroher U, Tipples GA, Tyler S, Vogrig R, Ward D, Watson B, Brunham RC, Krajden M, Petric M, Skowronski DM, Upton C, Roper RL: The Genome sequence of the SARS-associated coronavirus.Science 2003, 300:1399-1404. 10.1126/science.1085953
    CAS Article PubMed Google Scholar
  3. 3.
    Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, Penaranda S, Bankamp B, Maher K, Chen MH, Tong S, Tamin A, Lowe L, Frace M, DeRisi JL, Chen Q, Wang D, Erdman DD, Peret TC, Burns C, Ksiazek TG, Rollin PE, Sanchez A, Liffick S, Holloway B, Limor J, McCaustland K, Olsen Rasmussen M, Fouchier R, Gunther S, Osterhaus AS, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ:Characterization of a novel coronavirus associated with severe acute respiratory syndrome.Science2003, 300: 1394-1399. 10.1126/science.1085952
    CAS Article PubMed Google Scholar
  4. 4.
    Ng ML, Tan SH, See EE, Ooi EE, Ling AE:Proliferative growth of SARS coronavirus in Vero E6 cells. J Gen Virol 2003, 84:3291-3303. 10.1099/vir.0.19505-0
    CAS Article PubMed Google Scholar
  5. 5.
    Li M, Moore WJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC, Choe H, Farzan M:Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.Nature 2003, 426:450-454. 10.1038/nature02145
    CAS Article PubMed Google Scholar
  6. 6.
    Bergeron E, Vincent MJ, Wickham L, Hamelin J, Basak A, Nichol ST, Chrétien M, NG Seidah:Implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus.Biochem Biophys Res Comm 2005,326: 554-563. 10.1016/j.bbrc.2004.11.063
    CAS Article PubMed Google Scholar
  7. 7.
    Zhang Y, Li T, Fu L, Yu C, Li Y, Xu X, Wang Y, Ning H, Zhang S, Chen W, Babiuk LA, Chang Z: Silencing SARS-CoV spike protein expression in cultured cells by RNA interference.FEBS Lett 2004,560: 141-146. 10.1016/S0014-5793(04)00087-0
    CAS Article PubMed Google Scholar
  8. 8.
    Subbarao K, McAuliffe J, Vogel L, Fahle G, Fischer S, Tatti K, Packard M, Shieh WJ, Zaki S, Murphy B: Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice. J Virol 2004, 78:3572-3577. 10.1128/JVI.78.7.3572-3577.2004
    PubMed Central CAS Article PubMedGoogle Scholar
  9. 9.
    Yang ZY, Kong WP, Huang Y, Roberts A, Murphy BR, Subbarao K, Nabel GJ: A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature2004,428: 561-564. 10.1038/nature02463
    CAS Article PubMed Google Scholar
  10. 10.
    Bisht H, Roberts A, Vogel L, Bukreyev A, Collins PL, Murphy BR, Subbarao K, Moss B: Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice.Proc Natl Acad Sci USA 2004,101:6641-6646. 10.1073/pnas.0401939101
    PubMed Central CAS Article PubMedGoogle Scholar
  11. 11.
    Bukreyev A, Lamirande EW, Buchholz UJ, Vogel LN, Elkins WR, St. Claire M, Murphy BR, Subbarao K, Collins PL:Mucosal immunization of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet2004,363: 2122-2127. 10.1016/S0140-6736(04)16501-X
    CAS Article PubMed Google Scholar
  12. 12.
    Sainz B Jr, Mossel EC, Peters CJ, Garry RF:Interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (SARS-CoV).Virology2004, 329:11-17. 10.1016/j.virol.2004.08.011
    CAS Article PubMed Google Scholar
  13. 13.
    Stroher U, DiCaro A, Li Y, Strong JE, Aoki F, Plummer F, Jones SM, Feldmann H:Severe acute respiratory syndrome-related coronavirus is inhibited by interferon- alpha.J Infect Dis 2004,189:1164-1167. 10.1086/382597
    Article PubMed Google Scholar
  14. 14.
    Sui J, Li W, Murakami A, Tamin A, Matthews LJ, Wong SK, Moore MJ, Tallarico AS, Olurinde M, Choe H, Anderson LJ, Bellini WJ, Farzan M, Marasco WA:Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci USA2004, 101: 2536-2541. 10.1073/pnas.0307140101
    PubMed Central CAS Article PubMedGoogle Scholar
  15. 15.
    Savarino A, Boelaert JR, Cassone A, Majori G, Cauda R: Effects of chloroquine on viral infections: an old drug against today’s diseases? Lancet Infect Dis 2003, 3:722-727. 10.1016/S1473-3099(03)00806-5
    CAS Article PubMed Google Scholar
  16. 16.
    Ng ML, Tan SH, See EE, Ooi EE, Ling AE:Early events of SARS coronavirus infection in vero cells. J Med Virol2003, 71: 323-331. 10.1002/jmv.10499
    CAS Article PubMed Google Scholar
  17. 17.
    Simmons G, Reeves JD, Rennekamp AJ, Amberg SM, Piefer AJ, Bates P:Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry.Proc Natl Acad Sci USA 2004,101: 4240-4245. 10.1073/pnas.0306446101
    PubMed Central CAS Article PubMedGoogle Scholar
  18. 18.
    Yang ZY, Huang Y, Ganesh L, Leung K, Kong WP, Schwartz O, Subbarao K, Nabel GJ: pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN. J Virol2004, 78: 5642-5650. 10.1128/JVI.78.11.5642-5650.2004
    PubMed Central CAS Article PubMedGoogle Scholar
  19. 19.
    Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ: A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase.J Biol Chem2000,275: 33238-33243. 10.1074/jbc.M002615200
    CAS Article PubMed Google Scholar
  20. 20.
    Song HC, Seo MY, Stadler K, Yoo BJ, Choo QL, Coates SR, Uematsu Y, Harada T, Greer CE, Polo JM, Pileri P, Eickmann M, Rappuoli R, Abrignani S, Houghton M, Han JH: Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein. J Virol2004, 78:10328-10335. 10.1128/JVI.78.19.10328-10335.2004
    PubMed Central CAS Article PubMedGoogle Scholar
  21. 21.
    Keyaerts E, Vijgen L, Maes P, Neyts J, Ranst MV: In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine.Biochem Biophys Res Commun 2004,323: 264-268. 10.1016/j.bbrc.2004.08.085
    CAS Article PubMed Google Scholar
  22. 22.
    Thorens B, Vassalli P: Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion. Nature1986, 321: 618-620. 10.1038/321618a0
    CAS Article PubMed Google Scholar
  23. 23.
    Dille BJ, Johnson TC: Inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine. J Gen Virol 1982, 62: 91-103.
    CAS Article PubMed Google Scholar
  24. 24.
    Tsai WP, Nara PL, Kung HF, Oroszlan S:Inhibition of human immunodeficiency virus infectivity by chloroquine.AIDS Res Hum Retroviruses 1990,6: 481-489.
    CAS Article PubMed Google Scholar
  25. 25.
    Savarino A, Lucia MB, Rastrelli E, Rutella S, Golotta C, Morra E, Tamburrini E, Perno CF, Boelaert JR, Sperber K, Cauda RC: Anti-HIV effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors. J Acquir Immune Defic Syndr 2004,35: 223-232.
    CAS Article PubMed Google Scholar
  26. 26.
    Ducharme J, Farinotti R:Clinical pharmacokinetics and metabolism of chloroquine. Focus on recent advancements.Clin Pharmacokinet1996, 31: 257-274.
    CAS Article PubMed Google Scholar
Download references

Acknowledgements​

We thank Claudia Chesley and Jonathan Towner for critical reading of the manuscript. This work was supported by a Canadian PENCE grant (T3), CIHR group grant #MGC 64518, and CIHR grant #MGP-44363 (to NGS).

Author information​

Affiliations​

  1. Special Pathogens Brach, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, 1600 Clifton Road, 30333, USA
    Martin J Vincent, Bobbie R Erickson, Pierre E Rollin, Thomas G Ksiazek & Stuart T Nichol
  2. Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, 110 Pine Ave West, QCH2W1R7, Canada
    Eric Bergeron, Suzanne Benjannet & Nabil G Seidah

Corresponding author​

Correspondence toStuart T Nichol.

Additional information​

Competing interests​

The author(s) declare that they have no competing interests.

Authors’ contributions​

MV did all the experiments pertaining to SARS CoV infection and coordinated the drafting of the manuscript. EB and SB performed experiments on ACE2 biosynthesis and FACS analysis. BE performed data acquisition from the immunofluorescence experiments. PR and TK provided critical reagents and revised the manuscript critically. NS and SN along with MV and EB participated in the planning of the experiments, review and interpretation of data and critical review of the manuscript. All authors read and approved the content of the manuscript.

Authors’ original submitted files for images​

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1​

Authors’ original file for figure 2​

Authors’ original file for figure 3​

Authors’ original file for figure 4​

Authors’ original file for figure 5​

Authors’ original file for figure 6​

Rights and permissions​

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License (Creative Commons — Attribution 2.0 Generic — CC BY 2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Reprints and Permissions

About this article​

Cite this article​

Vincent, M.J., Bergeron, E., Benjannet, S. et al.Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J 2, 69 (2005). Chloroquine is a potent inhibitor of SARS coronavirus infection and spread - Virology Journal
Download citation

Share this article​

Anyone you share the following link with will be able to read this content:



Provided by the Springer Nature SharedIt content-sharing initiative

Keywords​

  • severe acute respiratory syndrome coronavirus
  • chloroquine
  • inhibition
  • therapy


Download PDF

logo.svg

Virology Journal​

ISSN: 1743-422X

1626100689785.png
 
Pythagoras said:
ni mbulula tu mwenye chuki binafsi na wazungu
Click to expand...
Ana chuki binafsi na mzungu wakati anatumia smartphone na hata internet ya mzungu.

Kasheheni machanjo ya mzungu - Zanzibar wameshaanza kuchanjwa kasikia nani kaganda damu?

Yuko kama yule jamaa wa kyoto tu.
 
brazaj said:
Ana chuki binafsi na mzungu wakati anatumia smartphone na hata internet ya mzungu.

Kasheheni machanjo ya mzungu - Zanzibar wameshaanza kuchanjwa kasikia nani kaganda damu?

Yuko kama yule jamaa wa kyoto tu.
Click to expand...
Washenzi tu na ushamba wao. Mimi mwenyewe hapa Tz nimechanja toka Juni 10. Na sina tatizo lolote. Wapuuzi hawa wa mwendazake
 

Updates:​

​

URGENT! Spanish researchers disclose ‘real’ cause’ of Covid-19 + Leaked Document + AAPS​

Posted on July 12, 2021 byState of the Nation
FacebookTwitterPinterestRedditEmail

Share

La Quinta Columna urgent announcement​

Our Greater Destiny
Thanks to Kathy
Spanish biostatistician Ricardo Delgado, Dr. José Luis Sevillano and the team of researchers and professors with whom they have been conducting their research confirmed the presence of graphene oxide nanoparticles in vaccination vials.
It has also been found on the swabs and on face masks.
Graphene Oxide is a toxic bioweapon and the real cause of COVID-19.

Before you vote read this​

Bioweapons can come in many forms, and this is the new tactic of war against the people by this and other governments. The powerful controlling element of society and its corrupt government partners care nothing about you or your families, but only about power and control over you. The real bioweapon is not any ‘virus,’ but is the ‘vaccine’ delivery system itself, along with masks, and testing, as perpetrated by the very entity (government) claiming to be your savior.Is Graphene Oxide Causing What Is Falsely Being Referred to as ‘Covid-19?’ - LewRockwell

LEAKED DOCUMENT – ‘AUTHORIZATION TO ADMINISTER A POISON (COVID-19) VACCINE’​

Support documents below video [6:20 mins]

Public Health Act 2016 (WA) – Instrument of Authorisation – Authorisation to Supply or Administer a Poison [SARS-COV-2 (COVID-19) VACCINE – Australian Defence Force] (No.2) 2021​

Guidance: An authorisation by the Chief Health Office under the s. 197 and s.198 Public Health Act 2016 (WA) to authorise relevant Australian Defence Force employees to supply and administer the COVID-19 Vaccine.
Public Health Act 2016 (WA) – Instrument of Authorisation – Authorisation to Supply or Administer a Poison [SARS-COV-2 (COVID-19) VACCINE – Australian Defence Force] (No.2) 2021
Poisons Standard June 2021
SARS-COV-2 (COVID-19) VACCINE Schedule 4 [Prescription Only Medicine, or Prescription Animal Remedy – Substances, the use or supply of which should be by or on the order of persons permitted by State or Territory legislation to prescribe and should be available from a pharmacist on prescription.]
https://www.legislation.gov.au/Details/F2021L00650
Part 3Miscellaneous Regulations, Section 1Advertising General 3
(1) A person must not include any reference to a poison included in:
b) Schedule 4 or Schedule 8,
of this Standard in any advertisement except in genuine professional or trade journals or other publications intended for circulation only within the medical, nursing, veterinary, dental or pharmaceutical professions or the wholesale drug industry.https://www.legislation.gov.au/Details/F2021L00650/Html/Text#_Toc72922496
I could not find SARS-COV-2 classified as a poison on the Consent form.https://www.health.gov.au/sites/def...vaccination-consent-form-print-friendly_0.pdf
Is ‘poison’ disclosed on COVID-19 Vaccine AstraZeneca or Comirnaty (Pfizer) information sheets to comply with full disclosure and informed consent?
PDF: Lists who is qualified to administer, their function, and conditions. Do administrators confirm full disclosure and informed consent?
https://www.wa.gov.au/sites/default...ister-COVID-vaccine-No2-Aus-Defence-Force.pdf
Five Eyes is the world’s oldest intelligence network and arguably the world’s most functional security partnership. Most commonly, the term refers to a unique signals intelligence pooling club of agencies from Australia, Canada, New Zealand, the United Kingdom and the United States. But FVEY, as it is also known, also refers to a large and growing number of special relationships and trans-governmental policy networks that bind these five states in virtually all areas of security.

What do doctors think about the COVID jab?​

By Jane M. Orient, M.D.
Executive Director
Association of American Physicians and Surgeons
Many patients tell us their doctors are pressuring them to get the COVID jab. The American Medical Association (AMA) claims 96 % of doctors are themselves fully vaccinated.
The Association of American Physicians and Surgeons (AAPS) decided to check out the AMA’s 96-percent claim. We recall that the “one voice” has sometimes been wrong, and the 4 percent right.
It turns out the AMA’s 300 survey respondents were not inclusive of all doctors. In the AAPS survey, nearly 60 % of some 700 respondents said they were NOT “fully vaccinated.” Of these, 80 percent said “I believe risk of shots outweighs risk of disease.”Read more at
https://highlandcountypress.com/Con...doctors-think-about-the-COVID-jab-/4/22/69691

Make it known​

Fact based evidence needs to be put in front of investigative journalists and activists, governments at all levels, employers, family doctors, other health providers, everyone pushing the jab and covid restrictions.
Withdraw consent by spreading evidence of crimes against humanity to end the fake flu coup, save the children, their family, and our species. TY
___
https://ourgreaterdestiny.org/2021/...e-real-cause-of-covid19-leaked-document-aaps/
 
You must log in or register to reply here.

Our Community

Regional Communities

Back
Top Bottom
Menu
Forums
New posts
Log in
Register