Are the COVID-19 Test Kits Designed to Produce False Positives (Plandemic)?

gl69m

Member
Post #29 (from original thread, from gl69m)
Post #29


Looked at several articles about viruses and coronaviruses (and 'sars-cov2' also) in particular and their potential viability to stay infective within wastewater/sewage. Not going to try and get too technical here, just comparing two recent articles alleging 'covid-19' ('sars-cov2' virus) found in high concentrations in waste water (and so alleging undetected {“untested' pcr I presume} infections), with older scientific journal articles studying viruses (and other coronaviruses in one of the articles) in wastewater/sewage.


Erie County sewage shows huge spike in COVID-19
June 23, 2020
https://www.goerie.com/news/20200623...ke-in-covid-19


Researchers track high levels of COVID-19 in Florida wastewater
Published: May 24, 2020
https://www.wtsp.com/article/news/he...d-d301d8c7f9cc


Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks
2014 Nov
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249052/


Survival of Coronaviruses in Water and Wastewater

Published online 2008 Dec 3
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091381/


Excretion and detection of SARS coronavirus and its nucleic acid from digestive system

2005 Jul 28
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434667/


First article, a Facebook friend acquaintance on my friend list, guy I went to highschool with in Collinsville IL, graduating class of 1987, he posted this article on FB more than a month ago, I'm just now getting around to a more detailed look at it, lazy me
biggrin.gif
. Me and this guy sparred a few rounds on Facebook about 'covid' and at least IMO of course I KO'd his stance several times, and especially when it came to the 'covid' vaccines and nano-technology, schooled him and his FB buddies ganging up on me in the comments of one of his posts. I won't name him, he is a herpetology proffessor, he's a smart guy I will admit, of course I am biased about what I believe about the 'covid' and he buys the 'covid' as though it was real although I know I put a serious dent in his certainty about it, but not likely will he admit to that; not worried about that though , he is still someone I can be relatively respectful with even in an intellectual confrontation so to speak.


Without further ado, the article he posted,

Researchers track high levels of COVID-19 in Florida wastewater
Published: May 24, 2020
https://www.wtsp.com/article/news/he...d-d301d8c7f9cc



Quote:


South Florida sewage is "chock-full" of the virus.

TAMPA, Fla. — It’s an overcast, but beautiful Sunday evening near Ben T. Davis Beach along the Courtney Campbell Causeway. People are out enjoying a nice swim and break from the heat.

But some researchers now want to know if bodies of water, like Tampa Bay, and others around the state could potentially contain dangerous levels of COVID-19 coronavirus after accidental sewage spills.

“Here in Miami-Dade, they’ve been doing some studies of the saltwater on the beaches to make sure they’re not contaminated,” said Dr. Aileen Marty, an infectious disease expert at Florida International University.

She, and other researchers around the county are keeping a close eye on what happens to Florida’s raw sewage and where it ends up.

“Our wastewater is chock-full of the virus,” said Marty, who has been looking at concentrations of the virus in sewage since Miami-Dade began taking samples in March. Ever since then, scientists have been working to estimate how many people are sick based on the concentration of virus in the wastewater.

This is a very short article with no details of the sample testing for viruses they did, nor how they came up with this estimate of possible 'covid-19' infections within this county (Hillsborough County, ~1.47 million population{2019}),

Quote:


They reported theirhighest concentration April 9, estimating 2 percent of the county’s population, or around 46,000 people, were infected.

I will of course quibble the shit out of this number, never mind for the moment that 'covid-19' ('sars 2.0' virus) isn't even a real virus (in the population now), supposing hypothetically another real coronavirus was currently in actual “pandemic”, would the undetected 'infection' (or supposed 'asymptomatic' but still alleged infections) number still really be so high if we took numbers reported akin to the numbers we now see according to the 'covid' narrative at face value? as a true infection rate (of at least some virus or other)? I have my doubts.


An article on enteroviruses in wastewater may shed light on that answer, will show that next.

Quote:


“As long as we have COVID circulating in our population and people using the sewer systems to relieve themselves, that wastewater is going to have virus in it,” Marty said.
Marty is increasingly worried about the potential for broken sewer lines, or even overflow during major rain events like hurricanes. The city of St. Petersburg previously struggled with multiple accidental releases of raw sewage or partially treated wastewater.
Swimming areas like Ben T. Davis Beach sometimes even have to be shut down when the local health department finds high levels of fecal bacteria.

Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks
2014 Nov
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249052/

Quote:


Abstract

Most persons infected with enterically transmitted viruses shed large amounts of virus in feces for days or weeks, both before and after onset of symptoms. Therefore, viruses causing gastroenteritis may be detected in wastewater, even if only a few persons are infected. In this study, the presence of eight pathogenic viruses (norovirus, astrovirus, rotavirus, adenovirus, Aichi virus, parechovirus, hepatitis A virus [HAV], and hepatitis E virus) was investigated in sewage to explore whether their identification could be used as an early warning of outbreaks. Samples of the untreated sewage were collected in proportion to flow at Ryaverket, Gothenburg, Sweden. Daily samples collected during every second week between January and May 2013 were pooled and analyzed for detection of viruses by concentration through adsorption to milk proteins and PCR. The largest amount of noroviruses was detected in sewage 2 to 3 weeks before most patients were diagnosed with this infection in Gothenburg. The other viruses were detected at lower levels. HAV was detected between weeks 5 and 13, and partial sequencing of the structural VP1protein identified three different strains. Two strains were involved in an ongoing outbreak in Scandinavia and were also identified in samples from patients with acute hepatitis A in Gothenburg during spring of 2013. The third strain was unique and was not detected in any patient sample. The method used may thus be a tool to detect incipient outbreaks of these viruses and provide early warning before the causative pathogens have been recognized in health care.


So this study from Sweden shows that “detection' of some of the viruses by the methods used appears to be pretty high in the untreated wastewater. Skimming thru the article it appears that the main “detection' method is pcr (real time “quantitative' I believe), I think they may have done one experiment to test actual infective recovery of actual viruses (microbiological technique generally done that is known as the “plaque” method, seeing how many “plaque” holes are formed in a lawn of cultured host cells {for the recovered virus particles to infect} on petri dishes).

The one particular result in Figure 2 highlights for me a rather large discrepancy with the alleged “viral genome recovery” rate found by the pcr results and correlating that to estimated 'infection rate' within the geographical location of the wastewater sampled and tested; although in their discussion I don't think they framed it as being a “large discrepancy” but I think that's what I would call it.

Not going to get too technical but I need some detail to compare the scientific accumen (accuracy and legitimacy) of this study with the 'covid-19' studies to see how much legitimacy (at least numerical wise of estimated 'infection rate') they have by comparison, so it's unfortunately necessary. And for the purpose of brevity and simplicity I will focus mostly only on the norovirus GII from this study.

Quote:


MATERIALS AND METHODS

Sewage treatment plant and sampling.


Sewage samples were collected at the wastewater treatment plant Ryaverket (owned and run by Gryaab AB), Gothenburg, Sweden. The plant receives and treats wastewater from a population of 690,000 in the Gothenburg region and industrial wastewater and storm water. While the inflow of wastewater from households is fairly constant during the year, the total flow increases during periods of heavy rain due the inflow of storm water. Such daily variations in the flow during the sampling period are shown in Fig. 1.
(not worried about figure 1)

Virus concentration.

The concentration of viruses from the weekly sample of wastewater was performed by virus adsorption to milk powder, mainly as previously described (29). Briefly, viruses in 1,000 ml sewage were adsorbed to 10 g acidified skim milk powder (pH 3.5 [Difco]) by stirring at room temperature for 8 h.
(not really worried about the whole viral recovery method here, and skipping the pcr viral genome detection method explanation too)
Calculation of the number of viral genomes and the approximate number of infected individuals shedding 1011 virus particles per day required for detection of viral genomes in sewage.

The CT values obtained for sewage samples in the qPCR were used to calculate the number of viral genomes (Ci) by performing linear regression of the CT values obtained from serial dilutions of pUC57cl in relation to the dilutions of the plasmid.


An infected individual is assumed to excrete between 107 and 1011 norovirus, HAV, enterovirus, and adenovirus particles per day (19).
Calculations of the number of infected persons shedding viruses in this study were based on the maximum amount of virus that is shed per day by a newly infected person, 1011 virus particles/day, thereby estimating the lowest number of individuals shedding virus into the wastewater.
The number of virus particles that was expected to be present in the sewage on the daily basis from one infected person excreting (Cexp) was calculated for each week according to the following equation: Cexp = 1011/{[Σ(respective daily flow)]/7}.

The number of potentially infected individuals (Ninfected) based on the presence of the respective virus in the sewage was estimated as Ninfected = Ci/Cexp.

Not gonna worry about the calculations and virus concentrations a whole lot here, this is only to compare methodology with the Erie PA paper. In figure 2 (derived from values in Table 2) it has a graph of “number of estimated individuals shedding norovirus GGII based on number of viral genomes identified in sewage” plotted with increment line on the left and this is compared to the “number of patients diagnosed with norovirus GGII”

Quote:


zam9991057710002.jpg


So the graph and the numbers derived from Table 2 shows that on week 7 the number of viral genomes detected by the pcr methodology from the pooled weekly sample of wastewater gives an expected number of ~1200 estimated infected persons shedding ~10¹¹ viral particles per day; but also shows only 44 diagnosed infections (from 135 tested I assume, or 44/135x100= lucky 33% positive) in that week of sample collection. But assume probable lag time for initial infection to symptom onset and diagnosis then for week 9 it shows 64/156 or 41% positive from those tested; and thus even if we add those together 44+64=108/1200 (x100) => then only a 9% positive rate of the number of diagnosed infections compared to the number expected infections base on the amount of viral genome (estimated number of genomes thus meaning viral particles) RNA supposedly recovered from the sewage. That seems like a pretty large discrepancy to me within this particular area of population.

Table 2 (first 5 viruses), https://www.ncbi.nlm.nih.gov/pmc/art...ort=objectonly

Here is this article's possible explanation as to why so few diagnosed infections compared to amount of RNA detected (and estimated virus concentration correlating to estimated “infections');

Quote:


DISCUSSION

Most studies on enteric viruses in sewage only detect the viruses and do not relate the virus sequences identified in sewage to those from patients from the same sampling time and region (27, 35,42).

There was a peak in number of detected viral genomes during week 7 for most of the investigated viruses in this study. This was the week for winter school holidays in this region of Sweden. The assays to detect the different virus types were not performed on the same day or even during the same week, which means that the identified elevated amount of virus particles in the wastewater probably did not reflect a systematic technical error but may indicate that there was an influx of persons from other regions who shed virus during these holidays.

I'm kind of assuming the explanation for the discrepancy is that an extra number of “infected' individuals who were traveling to Gothenburg around the holiday week shed the virus down the toilets into the sewers that was detected and these people must not have been diagnosed at least not diagnosed with these viruses (norovirus GII in particular in the example from Figure 2) in Gothenburg hospitals during those weeks. An extra estimated 1092 people I guess. Possible? I suppose but I feel some skepticism of that is highly warranted.


What about Astrovirus? According to week 7 they estimated 1720 infected persons but diagnosed only 0/135 (week 7) and 4/156 (or 2.4% positive), but out of 1720 expected “infections' only a 0.23% positive rate within Gothenburg based on number of genomes (virus particles) supposedly detected, and an even quite higher discrepancy in my view. Where are these supposedly “infected' people coming from (going to Gothenburg), from Stockholm or somewhere?

Is there other possible explanations for such high viral load in the sewage when there are so few diagnosed infections compared to expected estimated infection numbers? What came to my mind was is the pcr method over estimating the number of viral genomes, are they screwing it up or is the method incorrectly amplifying the real number of genomes (correlated 1 to 1 with one viral particle), or are they deliberately falsifying the expected numbers to get more health care funding or some other reason? These possibilities wouldn't be easy to find information to confirm or dis-confirm that's for sure.

Another thought I had was, since viruses can collect and precipitate with solid matter particulate in the wastewater and some of that particulate is organic matter, perhaps human and animal cells cells shed into the wastewater also, is it possible viral replication can occur within the wastewater itself and amplifying the total number of viruses per unit of wastewater (per liter or milliliter tested etc.)? Without digging through more articles I really don't know, I would think that this could be possible but I'm not going to say it is with any certainty as for now.

Another thought is perhaps the pcr method in this testing is really detecting a high rate of false positives, the primers used for each virus may simply be detecting non related genetic material, but without seeing validation numbers (false pos. rate %) for each primer/pcr method for each virus cannot say for sure how many false positives could be expected, but we should also keep in mind that the extreme organic soup of the sewer water of course, so has to be a huge amount of microbes of all kinds and tons of genetic material (DNA/RNA) of all kinds that is probably not really filtered out (or not a whole lot I suspect) by the extraction/concentration methods which logically I think could simply be contamination of genetic material that may interfere with trying to detect one specific viral RNA genome section per test.

One thing I find slightly strange in Table 2 is that the number of “diagnosed/tested” for each row (No. of persons) for the first 5 viruses (Norovirus GI, Norovirus GII, Astrovirus, Adenovirus, Rotavirus), the number of “tested” is the same number for each week (the columns at top of the table, “Parameter and virus” “Result for sampling wk:”)- week 3 (90), week 5 (122), week 7 (135), week 9 (156), week 11 (159), week 13 (166), week 15 (142). So for instance I focused on week 7, 4 of these viruses (Norovirus GI is an exception in that it is showing no tested/diagnosed, as 0 for weeks 3-9) show the highest or very high genome (alleged virus) concentrations on week 7, and that number tested for each of these 4 viruses is 135; does that mean that there were 135 people tested for all 4 of those viruses at once and each week also? If that is the case and I am not mistaken that would seem to mean that it might be possible some of these who tested positive for any of these may have tested positive for more than one virus? But it doesn't give enough information to definitively conclude that of course, I just found that curious, surely it does mean that these people were all tested for each virus. Perhaps their “specimens” are tested with a enterovirus panel containing primers for all of these, whether in one sample tested or they take multiple samples extracted from each specimen etc., not really sure but I think that might be the case.

So the article concludes basically this type of methodology can be used to possibly monitor “estimated' infection/epidemiology rates of a population from viral testing in wastewaters, which I feel this study would tend to bolster studies now being conducted now of “testing' for 'covid-19' virus as likely being valid (in that such methodology of testing wastewater for viruses before 'covid' has at least been used before):

from the Discussion section,

Quote:


Analysis of wastewater for enterically transmitted viruses has a number of advantages as it makes it possible to monitor a large population by analyzing samples collected in one place, since wastewater from hundreds of thousands of households gathers at the same wastewater treatment plant. Wastewater contains enteric viruses excreted from persons who are ill as well as from cases of subclinical infection, which reflects the real magnitude of circulating virus in the community. In addition, virus can be detected before an outbreak occurs, as shown in this study for norovirus, since the virus may be excreted in feces before onset of symptoms (50), which is 1 to 2 days for norovirus GII and 4 to 5 days for astrovirus, (51), while for hepatitis A and E viruses, the excretion period is longer and occurs for up to 7 weeks (8, 52).


Detection of norovirus in sewage has been extensively studied, since it is known to cause large waterborne and seasonal outbreaks, sometimes with severe consequences for vulnerable persons (3, 26, 53,57). In this study, the amount of norovirus GII in wastewater peaked 2 to 3 weeks before the infection spread in hospital wards and nursing homes, indicating that the outbreak was ongoing at least 2 weeks before most persons severely affected by the norovirus infection came to medical attention.

Genetic comparison of viruses in sewage with virus from clinical sporadic cases and from outbreaks may provide a model for understanding the epidemiology of enteric viral pathogens in the population.


So let's go ahead and look at the Erie County sewage article, Rob (Truthissweet) sent me this article a few weeks ago (I think), and so I wanted to compare this to the other articles and I have been slow getting to it.

Erie County sewage shows huge spike in COVID-19
June 23, 2020
https://www.goerie.com/news/20200623...ke-in-covid-19

Quote:


Paul Totleben collecrting samples on May 21.

Howard Nadworny, M.D.: “These numbers make me worried.”
The first batch of results from a new wastewater testing program suggest that the reach of COVID-19 in Erie County has been far more widespread than previously known.

The most recent sewage testing estimated more than 20,000 cases of the virus in Erie County in early June — a figure far higher than the tally reached through standard COVID-19 testing.

Howard Nadworny, M.D., a Saint Vincent Hospital infectious diseases specialist and volunteer adviser to the county health department, thinks that estimate could be higher than the true total.

But the results are concerning, he said, and show that Erie County has seen a huge spike in the virus in the past month.

“I think what the sewage testing really shows us is that there’s been a rapid increase in the amount of virus in Erie County, which would predict that we’re going to have a lot more cases if people don’t do the few things that work,” like wearing face masks and maintaining social distancing, he said.

“These numbers make me worried,” he said.

The city of Erie’s Bureau of Sewers began taking samples of the county’s wastewater in late May, in hopes that the results, provided by a Massachusetts-based company called Biobot, could help identify COVID-19 outbreaks early.

The testing works by checking for fragments of genetic material from SARS-CoV-2, the virus that causes COVID-19. People who have the virus can shed these fragments in their stool, even if they are not showing symptoms, making sewage a potential tool in the fight against the disease.
Nadworny provided the first set of Biobot results to the Erie Times-News on Tuesday. The set showed results from wastewater samples taken on May 20, May 26, June 1 and June 8.

All four samples showed concentrations of SARS-CoV-2, which is measured in the number of “copies” of the virus detected per liter of sewage.
By dividing the number of copies by the approximate amount of the virus that each infected person is believed to shed, Biobot can reach an estimate for the total number of cases in a community, Nadworny said.
The May 20 results showed about 9,500 copies of the virus per liter. Biobot estimated the total number of COVID-19 cases in the community at 1,800 based on that finding.

At that time, Erie County was reporting a cumulative total of 163 cases based on testing of individuals.

There’s a difference in the results because we don’t have the ability to test people on a massive scale, Nadworny said. The sewage testing helps fill gaps in the data by offering a broad picture of the virus’s spread in the community.

The May 26 sample showed nearly 32,000 copies for an estimated 5,600 cases, and the June 1 sample showed 26,000 copies and 4,700 cases.

By June 8, the number of SARS-CoV-2 copies in the city’s June 8 wastewater sample had grown to more than 126,000.

Biobot estimated the total number of cases in Erie County at 20,800.
Preliminary results from the latest round of sampling, on June 15, show 141,000 copies of the virus. Nadworny expects the final results from that period will estimate Erie County has 24,000 cases of COVID-19.

Nadworny said he believes it will be relatively safe for businesses to reopen as Erie County enters the green phase of coronavirus restrictions on Friday, so long as masking requirements are enforced.

He’s more concerned about people who think the pandemic is over and they no longer have to take precautions.

Biotbot, a startup formed by researchers with the Massachusetts Institute of Technology, launched its sewage testing program in an effort to map the spread of COVID-19.
MIT”, same place that created the “quantum dot” vectors for the tattoo like vaccine injection patch that is likely going to be used for a 'covid' vaccine(s).
Erie County is one of 400 communities nationwide to participate in the Biobot testing, Nadworny said.

The Erie Community Foundation has issued a $30,000 grant that will cover continued sewage testing through the summer and fall, he said.

Well this article does not state any of the methodology used nor the pcr protocols used either (primers, other controls etc.), perhaps you'd have to contact somebody like the M.D. Nadworny or the lab that the Erie’s Bureau of Sewers sends the sample to etc., in my experience it seems very unlikely they would give it you if they even responded. For possible hypothetical sake, let's assume their virus capture/extraction/pcr detection methods are similar to the Swedish study, or may be similar to another coronavirus study I will examine next.

So as of May 20, they were saying these sewer results for 'testing' for 'sarscov2' 'genomes' yielded a possible 1800 cases but there were 163 diagnosed cumulative for that week (or for the whole pandemic to date for then?), so 163/1800 x 100 => ~9%, I'd say almost the exact same as we saw for norovirus GII in the Swedish study. Was there a butt-load of extra tourists at this time point shedding extra 'covid' virus into Erie's sewers- I would think surely not. Perhaps they will claim these are would be “asymptomatic' cases then (or maybe supposedly sick people not seeking treatment etc.), just simply not haven't been tested, perhaps that may also be said in the case of the enteroviruses in the Swedish study too, maybe, I don't recall seeing that as an explanation in that article though.
On PA's coronavirus page there is a pdf for “County case counts by date” link,

https://www.health.pa.gov/topics/Doc...s_7-7-2020.pdf

as of July 6th their 'counts' show 679 cases (585 'confirmed' and 94 'probable', 12,525 persons “negative' with pcr), so 679/24,000 x100=> ~2.8% positive out of expected, and 585/13,204=> ~4.4% positive rate out of those “tested'. If these tests have at least a 4% false positive rate (or more) than even if the virus was never even present it seems we would expect the ~4.4% positive rate anyways seems to me.

I notice in Philadelphia it shows a rate of 22,294/137,701x100=>~16.2% positive test rate and only 8 'probable' cases. That seems pretty high compared to a few other counties I looked at that were in the 5 to 6% range, and pretty low on the 'probable' category: perhaps this is because there is definitely has a higher black population/urban area, but I'd have to look at a lot more demographic numbers in a lot more counties and states before unequivocally declaring that to be true but I have seen quite a few articles stating that 'covid' is dis-proportionally “impacting' the black community especially so it is probably likely that examination would reveal that.

That is one worry for sure and we also need to concern age in understanding more dis-proportional “impact' as well: I estimate from the graph numbers by demographics that at least 25% of supposed 'covid' deaths in PA are black people (and blacks represent ~12% of population according to the demographics of PA), and on the age demographic around ~90% of 'covid' deaths are age 60 years and older (almost ~56% age 80 and older, to be expected for most such types of illness). (https://www.health.pa.gov/topics/dis...ges/Cases.aspx)

Let us go ahead and look at the next article for comparison here, one thing to note is, that only the 229E coronavirus was tested in this study and not the SARS CoV virus.

Survival of Coronaviruses in Water and Wastewater

Published online 2008 Dec 3
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091381/

Quote:


Abstract

The advent of severe acute respiratory syndrome and its potential environmental transmission indicates the need for more information on the survival of coronavirus in water and wastewater. The survival of representative coronaviruses, feline infectious peritonitis virus, and human coronavirus 229E was determined in filtered and unfiltered tap water (4 and 23°C) and wastewater (23°C). This was compared to poliovirus 1 under the same test conditions. Inactivation of coronaviruses in the test water was highly dependent on temperature, level of organic matter, and presence of antagonistic bacteria. The time required for the virus titer to decrease 99.9% (T99.9) shows that in tap water, coronaviruses are inactivated faster in water at 23°C (10 days) than in water at 4°C (>100 days). Coronaviruses die off rapidly in wastewater, with T99.9 values of between 2 and 4 days. Poliovirus survived longer than coronaviruses in all test waters, except the 4°C tap water.
Keywords: Survival, Water, Wastewater, Coronavirus, SARS
Introduction

The 2003 epidemic of severe acute respiratory syndrome (SARS) resulted in over 8,000 cases worldwide with a mortality rate of approximately 10% (Manocha et al. 2003; Centers for Disease Control 2004). The last known outbreak occurred in a research lab in Beijing in 2004. (I have a really hard time believing this, that there were no more supposed cases of this virus after 2004, and they worked a vaccine from roughly 2006 to 2018 or so and then stopped and never completed one according to certain articles, makes no fucking sense.)
The cause of this disease was identified as a novel human coronavirus with probable origins in civets and a possible reservoir in bats (Guan et al. 2003; Lau et al. 2005; Wang et al. 2006). Coronaviruses are enveloped, single-stranded RNA viruses that range from 60 to 220 nm in size. They can infect birds and mammals, including humans, and are transmitted through aerosols or the fecal-oral route. The rapid spread of coronaviruses during outbreaks suggests the primary mode of transmission of human coronaviruses is respiratory droplets; however, there is no direct evidence to support this (Belshe 1984). Since coronavirus infection in humans up to this point has been characterized as a mild, self-limiting condition, there is limited information on its transmission potential through the environment.
The SARS epidemic had potential links to water and wastewater given that the March 2003 outbreak at the high-rise housing estate in Hong Kong involving over 300 people was linked to a faulty sewage system (Peiris et al. 2003). The fact that SARS-CoV can replicate in the enteric tract (Leung et al. 2003) makes it a possible enteric pathogen, and the incidence of diarrhea ranging from 8 to 73% in SARS cases (SARS Epidemiology Working Group 2003) causes concern about its potential environmental transmission. Leung et al. (2003) also reported that viral cultures from SARS patients recovered higher yields from the small intestine than the lung tissues, which are the target organs of this virus. Infectious virus has been cultured from stools of SARS patients up to 3 weeks post infection (Chan et al. 2004; Liu et al. 2004)(this article https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323244/ shows that sars {original} virus was cultured in cell culture, and so far I have seen potentially only one or two articles showing any such evidence for 'sarscov2'). The advent of SARS and the question of its transmission indicate the need for more information, specifically the survival of coronavirus in water and wastewater. This study compared the survival of representative coronaviruses and poliovirus 1 in tap water and wastewater.
Materials and Methods (not worried so much about handling of the control viruses here in first paragraph, skipping to second paragraph)
Tap water samples were collected from a cold tap faucet in the laboratory.
Virus survival was determined in both nonfiltered tap water and tap water passed through a 0.2-μm pore size filter to remove bacteria. (I don't think this filtering was done in the Swedish study)
Tubes were sampled after 1, 3, 6, 10, 15, and 21 days and the samples frozen at −80°C until they were assayed on cells.

Samples of primary and activated sludge (secondary) effluent were collected in sterile polypropylene bottles from the Roger Road Wastewater Treatment Plant in Tucson, AZ, USA.
Samples were collected after 1, 2, 3, 6, 10, 15, and 21 days and the samples frozen at −80°C until assay.

Viruses were enumerated on cell cultures using either the plaque assay or TCID50 technique. PV-1 was titered in 6-well plastic cell culture plates by the plaque assay method (Payment and Trudel 1993). This is a direct quantitative method with a minimum detection limit of 10 pfu/ml. Each dilution was plated in duplicate wells. Coronaviruses, which do not form plaques in cell culture, were titered in 24-well plastic cell culture plates by the tissue culture infectious dose 50% technique (TCID50) (Payment and Trudel 1993). This technique determines the dilution at which 50% of the wells show CPE. Taking the inverse log of this dilution gives a titer of the virus per ml TCID50. The minimum detection for this method was 3.7 viruses per ml. Each dilution was plated in a minimum of 8 wells. Any samples that were not from test waters filtered prior to adding virus had to be filtered before assaying on cell culture to eliminate bacterial contamination. The 0.2 μm low protein binding Millex filters (Millipore, Billerica, MA) with polyethersulfone (PES) membrane were prepared by passing 3% beef extract (Becton Dickinson, Sparks, MD) at pH 7 through to block sites that might adsorb virus. All experiments were performed in triplicate.

159415f0bdba382eb1.png


Factors that can influence virus survival in water include temperature, organic matter, and aerobic microorganisms (John and Rose 2005; Melnick and Gerba 1980; Sobsey and Meschke 2003). The most critical influence on virus survival is temperature. It has been shown that virus survival decreases with increasing temperature, mainly caused by denaturation of proteins and increased activity of extracellular enzymes (Hurst et al. 1980; John and Rose 2005).

The increase in virus titer over time from the initial titer in the 4°C water can be attributed to the tendency of viruses to form aggregates that then disaggregate, not from viral replication in the sample. (this statement makes a blanket statement that does not look like they made any attempt to show that proven in this study or reference to other studies addressing this. Without further digging into scientific articles in reference to that question, I will defer to viral replication in wastewater as possible until I see good evidence it is proven otherwise.)
The presence of organic matter and suspended solids in water can provide protection for viruses that adsorb to these particles but at the same time can be a mechanism for removal of viruses if the solids settle out.
The titer of the coronaviruses immediately decreased 99.9% upon addition to the wastewater, while the PV-1 titer only dropped 10%. This decrease may be due to the presence of solvents and detergents in wastewater that would compromise the viral envelope and ultimately inactivate the virus. (this is a very important point considering all the attention that 'covid' has garnered with the soap and warm water and handwashing for a minimum of 20 seconds etc. for “inactivation” of the coronavirus.)
The presence of predatory microorganisms, such as protozoa, can increase the inactivation rate of virus in water, as well as the action of proteases and nucleases (Gerba et al. 1978; John and Rose 2005).

Conclusion

The results of this study indicate that coronaviruses are much more sensitive to temperature than PV-1 and that there is a considerable difference in survivability between PV-1 and the coronaviruses in wastewater.
This may be due in part to the fact that enveloped viruses are less stable in the environment than nonenveloped viruses. Coronaviruses die off very rapidly in wastewater, with a 99.9% reduction in 2–3 days, which is comparable to the data on SARS-CoV survival (Wang et al. 2005a, b).(I looked briefly at the SARS-CoV survival in wastewater article, I think it does show that SARS-CoV is similar to the 229E coronavirus as far as “inactivation” in the wastewater shown in this article, not going to look at that article in this post {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434667/}but I it does state that SARS-CoV RNA could be detected in sewage and stool samples but no infective virus detected in stool and sewage or urine samples. Another article I saw more recent than 2005 but before 2020 showed that allegedly “infective” SARS-CoV virus was found in {or cultured from} stool samples.) Survival of the coronaviruses in primary wastewater was only slightly longer than secondary wastewater, probably due to the higher level of suspended solids that offer protection from inactivation. PV-1 survived substantially longer than coronaviruses, requiring 10 days for a comparable reduction in primary wastewater and 5 days in secondary wastewater. This study demonstrates that the transmission of coronaviruses would be less than enteroviruses in the aqueous environment due to the fact that coronaviruses are more rapidly inactivated in water and wastewater at ambient temperatures./


So what we generally see from the two non-'covid' articles is that viruses are inactivated over time in wastewater and the inactivation increases with temperature, and that coronaviruses in general (229E and SARS-Cov) are inactivated quicker (because of their envelope) than non-envoloped (enteroviruses in this case) viruses; and that viral RNA “estimated' copy number (genome number and thus supposed “virus” number/concentration extrapolated from signal magnitude during pcr thermal cycling) seems to show a considerably higher viral concentration in the waste water than is commensurate with the detection(testing)/diagnosis of patient/cases in the same population area, for both other viruses and the 'covid' virus (reported this year). We saw a similar percentage of seeming over estimate of potential number of cases compared to reported case numbers from Erie county as compared to the enteroviruses in the Swedish study.

I will come back to look at the last article and (maybe more articles not sure yet) perhaps make this a part 2 post of this post.


Excretion and detection of SARS coronavirus and its nucleic acid from digestive system

2005 Jul 28
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434667/
 

gl69m

Member
Skipping ahead to Post #35 from the original thread, break down I did of this article here,
very pertinent

Post #35 (from gl69m)


Ok, so this article here purportedly provides evidence of 'sars-cov2' virus isolation, I am using selected to excerpts to cut down on total length of the posts etc,

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

Published: 03 February 2020

https://www.nature.com/articles/s41586-020-2012-7
Abstract

Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1,2,3,4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5,6,7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor—angiotensin converting enzyme II (ACE2)—as SARS-CoV.
Now my main thought has been for quite awhile now, that the genome presented as 'sars-cov2' is a mashup (fictional) from the original “sars-cov” virus and one of the bat coronaviruses. The very curious statement that the '2019-nCoV' virus (or viruses?) isolated from one of the 5 patients could be neutralized by sera from several patients, I'm wondering which patient's “sera” they used, so far I didn't see that question answered in this paper. Onto the “Main” body of the article,
Main

Here we report on a series of cases caused by an unidentified pneumonia disease outbreak in Wuhan, Hubei province, central China. This disease outbreak—which started from a local seafood market—
is associated with 80 deaths and has led to the infection of 33 people in 10 additional countries as of 26 January 202012.

Samples from seven patients with severe pneumonia (six of whom are sellers or deliverymen from the seafood market), who were admitted to the intensive care unit of Wuhan Jin Yin-Tan Hospital at the beginning of the outbreak, were sent to the laboratory at the Wuhan Institute of Virology (WIV) for the diagnosis of the causative pathogen (Extended Data Table 1). As a laboratory investigating CoV, we first used pan-CoV PCR primers to test these samples13, given that the outbreak occurred in winter and in a market—the same environment as SARS infections.
So samples from these seven patients are sent to WIV (Wuhan Institute of Virology) for diagnosis, and a set of “pan-CoV” PCR primers (“fishing” primers I'd call them) are used to test the samples, for possible coronavirus(es) (as in plural meaning screening for multiple different “coronaviruses” at the same time I'm assuming here); given that the outbreak is in the winter time there, “THE SAME ENVIRONMENT AS SARS INFECTIONS”.

Now I have repeatedly said here that it makes logical sense to me that the original SARS virus must still be circulating around (MERS virus too), despite some of these scientific articles I've seen make claims that the original SARS virus more or less disappeared somehow in late 2004 and no more (or maybe very very few perhaps claimed?) cases were seen after 2004. Is this paper showing a tacit admission from this Chinese study that the SARS virus is not only still around but is routinely screened for? basically like every winter flu/corona season and particularly in China since SARS originated there? And it is screened for with the “pan CoV” primers looking for it and or other common coronaviruses, and also looking for new coronavirus crossovers, from bats, civets, pangolins or camels or whatever animals all these different coronaviruses can infect. I would think so, would be logical in my view.

Part of my suspicion is that these PCR testing kits could also include extra primers not listed, in low enough concentration to not show if quality control tested for, but enough trace primers to potentially cause a false positive or occasionally catching some other coronavirus/common cold infections and not 'covid', okay just speculation on my part so what, everybody speculates sometimes, get off of me:(.
We found five samples to be PCR-positive for CoVs.One sample (WIV04), collected from the bronchoalveolar lavage fluid (BALF), was analysed by metagenomics analysis using next-generation sequencing to identify potential aetiological agents. Of the 10,038,758 total reads—of which 1,582 total reads were retained after filtering of reads from the human genome—1,378 (87.1%) sequences matched the sequence of SARSr-CoV (Fig. 1a). By de novo assembly and targeted PCR, we obtained a 29,891-base-pair CoV genome that shared 79.6% sequence identity to SARS-CoV BJ01 (GenBank accession number AY278488.2). High genome coverage was obtained by remapping the total reads to this genome (Extended Data Fig. 1). This sequence has been submitted to GISAID (https://www.gisaid.org/) (accession number EPI_ISL_402124). Following the name given by the World Health Organization (WHO), we tentatively call it novel coronavirus 2019 (2019-nCoV). Four more full-length genome sequences of 2019-nCoV (WIV02, WIV05, WIV06 and WIV07) (GISAID accession numbers EPI_ISL_402127–402130) that were more than 99.9% identical to each other were subsequently obtained from four additional patients using next-generation sequencing and PCR (Extended Data Table 2).
159415f277eff96e34.png

a, Metagenomics analysis of next-generation sequencing of BALF from patient ICU06.
I'm guessing by “SARSr-CoV” this means a generic SARS like coronaviruses, upon which the specific coronavirus belonging to the generic genetic reads (blocks of so many nucleotides in length etc., not fully sure how that is done/broken down) is identified with further more specific primer targeting, I guess.

So one sample, WIV04 was then used to further test and then flesh out the potential genome of the newly emerging 'discovered' '2019-nCoV' virus. Notice in Figure 1a the reads detected 6 total viruses apparently from this patient's sample (WIV04 is apparently patient ICU06, see Extended Data Table 1 {https://www.nature.com/articles/s41586-020-2012-7/tables/1}). So these other viruses, killer virus M1 (a virus infecting yeast), two different ichnoviruses, a mottle virus, and a Proteus phage VB; were apparently read from the “Metagenomics” analysis, does this mean that this patient is infected with 6 different viruses at the same time in her lungs?? Possibly, I guess?. Does this also mean that “pan-cov” fishing(my adjective) primers can catch a shit load of other sequences other than coronaviruses, I would have to assume so since the reads (more than 10 million) have to be filtered out for human and other microbe genomes in the meta-analysis, so surely the primers are “generic” as it were and not super specific only for a family of viruses let alone one specific virus. To me this would potentially call into question this methodology here to 'detect' a possible new virus and stitch a 'genome' together from all this genetic material being amplified to read and sequence from the generic “pan-CoV” primers, at least in my mind anyway.


https://www.nature.com/articles/s41586-020-2012-7/figures/1
Fig. 1: Genome characterization of 2019-nCoV.

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


a, Metagenomics analysis of next-generation sequencing of BALF from patient ICU06.
159415f277eff75a9b.png

b, Genomic organization of 2019-nCoV WIV04. M, membrane. c, Similarity plot based on the full-length genome sequence of 2019-nCoV WIV04. Full-length genome sequences of SARS-CoV BJ01, bat SARSr-CoV WIV1, bat coronavirus RaTG13 and ZC45 were used as reference sequences. d, Phylogenetic tree based on nucleotide sequences of complete genomes of coronaviruses. MHV, murine hepatitis virus; PEDV, porcine epidemic diarrhoea virus; TGEV, porcine transmissible gastroenteritis virus.The scale bars represent 0.1 substitutions per nucleotide position. Descriptions of the settings and software that was used are included in the Methods.

(Back to Main)

The virus genome consists of six major open-reading frames (ORFs) that are common to coronaviruses and a number of other accessory genes (Fig. 1b). Further analysis indicates that some of the 2019-nCoV genes shared less than 80% nucleotide sequence identity to SARS-CoV.However, the amino acid sequences of the seven conserved replicase domains in ORF1ab that were used for CoV species classification were 94.4% identical between 2019-nCoV and SARS-CoV, suggesting that the two viruses belong to the same species, SARSr-CoV.

We then found that a short region of RNA-dependent RNA polymerase (RdRp) from a bat coronavirus (BatCoV RaTG13)—which was previously detected in Rhinolophus affinis from Yunnan province—showed high sequence identity to 2019-nCoV. We carried out full-length sequencing on this RNA sample (GISAID accession number EPI_ISL_402131). Simplot analysis showed that 2019-nCoV was highly similar throughout the genome to RaTG13 (Fig. 1c), with an overall genome sequence identity of 96.2%.Using the aligned genome sequences of 2019-nCoV, RaTG13, SARS-CoV and previously reported bat SARSr-CoVs, no evidence for recombination events was detected in the genome of 2019-nCoV.
BALF= bronchial alveolar lavage fluid, BALF is collected by a procedure described as thus (google search):”Bronchoalveolar lavage (BAL) is a medical procedure in which a bronchoscope is passed through the mouth or nose into the lungs. Fluid is then squirted into a small part of the lung and then recollected for analysis.”. Squirt a little fluid into the lungs and then vacuum suction it back up to collect cellular debris including any germs/infection that are possibly present.

Phylogenetic analysis of the full-length genome and the gene sequences of RdRp and spike (S) showed that—for all sequences—RaTG13 is the closest relative of 2019-nCoV and they form a distinct lineage from other SARSr-CoVs (Fig. 1d and Extended Data Fig. 2). The receptor-binding spike protein encoded by the S gene was highly divergent from other CoVs (Extended Data Fig. 2), with less than 75% nucleotide sequence identity to all previously described SARSr-CoVs, except for a 93.1% nucleotide identity to RaTG13 (Extended Data Table 3). The S genes of 2019-nCoV and RaTG13 are longer than other SARSr-CoVs. The major differences in the sequence of the S gene of 2019-nCoV are the three short insertions in the N-terminal domain as well as changes in four out of five of the key residues in the receptor-binding motif compared with the sequence of SARS-CoV (Extended Data Fig. 3). Whether the insertions in the N-terminal domain of the S protein of 2019-nCoV confer sialic-acid-binding activity as it does in MERS-CoV needs to be further studied. The close phylogenetic relationship to RaTG13 provides evidence that 2019-nCoV may have originated in bats.
I'm pausing here to note that the genetic similarity/homology between the alleged discovered bat coronavirus dubbed “RaTG13” and “2019-nCoV”(renamed 'SARS-CoV-2') is 96.2% nucleotide for nucleotide.
(phylogenetic analysis of s gene (spike protein) and RdRp(RNA-dependent RNA polymerase), genetic relation between different coronaviruses based on just these two genes: )
159415f277effa9af5.png

159415f277eff9e764.png

a, b, Phylogenetic trees on the basis of the gene sequences of S (a) and RdRp (b) are shown. 2019-nCoV and bat CoV RaTG13 are shown in bold and in red. The trees were constructed using the maximum likelihood method using the GTR + G substitution model with bootstrap values determined by 1,000 replicates. Bootstraps values of more than 50% are shown.
I have no idea what “bootstrap” values are lol! Pull yourself up by the viral bootstraps haha!, not worried about that right now. SARS-CoV BJ01 seen above the red font 2019-nCoV and Bat Cov RaTG13 (genomes for these two genes)- BJ01 is the genome (relation) of the original (2002) SARS virus samples(“isolated”). “Common cold” human coronaviruses (OC43, NL63, 229E, HKU's) are seen below the red font ones, bat coronaviruses are mixed in above and below the red font ones.

I'm also gonna pause here to look at the comments on this paper posted below the article, very interesting and some strong negative critiques to the author(s)' work here, some relative skepticism I think too about how valid this work is in calling it an 'isolation' of 2019-nCov ('sars-cov-2'), at least in my opinion.

Comments:



  • Acron Naji4 days ago
    According to Shi's response to Science magazine, and her TV interview before, she doesn't have the isolated RaTG13 virus and any samples at her lab in Wuhan. This means there is no way to requecence RaTG13 scientifically and verify it existed or not in nature. Since it's crucial for the seeking of origin of SARS-Cov-2, I personally call for her retracting of this article.
    https://www.sciencemag.org/...

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    Alberto Maria Cattaneo17 days ago
    I also realized some inconsistency about the information available for RaTG13, have a look at my preprint on Researchgate: https://doi.org/10.13140/RG....
    Zhou et al. mention to have collected RaTG13 samples from bats of the Yunnan province on 2013, but checking on GISAID, and all the other databases, RaTG13 was deposited only on 2020, no evidences that it was really sampled 7 years ago are available, apart "saying so".
    Plus, in my preprint, I sequence-aligned the S-proteins of Covid-19 and RaTG13 with other Sars-Covs´, including Pangolin´s (Zhang et al. 2020 https://doi.org/10.1016/j.c... and I did not validated the same identity of the binding motif of the RBD that Zhang describes, neither I found evidences of the existence of a putative furin recognition motif in the S-proteins of Pangolin´s viruses.
    All together, this rise further doubts about the origins of RaTG13. Supplementary evidences about the Sars-CoV-2 origins from Zhou et al. would be really appreciated.

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    Monali C. Rahalkara month ago
    We have published a critical view on this paper in our preprint: https://www.preprints.org/m...

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    Samuel Soroaster3 months ago
    How could this ever get published in Nature? Did the reviewers not note that the most important reference namely the RATTG13 Sequence happend to be published by the same lab only a few days before publication? Why would they wait 7 years only until after the outbrake in Wuhan to publish the RATTG13 sequence? This needs to be followed up by the editor. https://www.ncbi.nlm.nih.go...

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    prajwol3 months ago
    I could not find accession numbers of reference sequence data that were used in phylogenetic analysis. If I were to replicate this study, how am I supposed to do so without any metadata table of the GenBank accession numbers of those reference sequences? How can Nature journal ignore these important component?


    Kenneth McDonald3 months ago • edited
    I just wrote this to Health Feedback.org
    QUOTE: "Covid-19 death predictions were wildly exaggerated by Imperial College. This helped to unreasonably form the idea lockdowns should occur in the Third World that will cause tens of millions of innocent women, children and babies to starve to death, in vastly higher numbers than the elderly might be saved to live a bit longer.
    I agree the article "Manufactured Pandemic" did not have enough supporting evidence in it to prove some of its claims. All that proves is the article was badly written, not that the PCR Test did not at first have inefficient primers, or other possible failures. Your "refutation article" is being used to block anyone wanting to prove that they CAN come up with evidence missing from the Manufactured Pandemic article, as if you disproved the test has flaws, when all you proved was the article never had proof links.
    Similarly Flora Teoh made the accusation the writer of the article was attacking Kary Mullis brilliant PCR technology, when all he/she was attacking was its particular use in this test. You then went on to say the person was "therefore" denying PCR can ever show viral load in OTHER tests or when he/she never. The ISSUE was did that specific test show viral load? As f it never we all have perhaps 10 viruses in our bodies in low viral load that do us no harm, (as Dr Andrew Kaufman points out) thus without viral load indication IN THAT ACTUAL TEST the test proves next to NOTHING.
    You then allow no comments.
    Do you realise that if you are saying the first 1 - 3 million stats are accurate when they are not that those stats (I believe are bogus) are being used to starve to death TENS OF MILLIONS of children in the Third World? AND your work is being used to stifle freedom of speech as well? Then you allow no comments to refute you?
    Please reply to this message."
    see more

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    Kenneth McDonald3 months ago • edited
    Why is it that only one scientist, Flora Teoh, steps forward and says the PCR Test used on the first 3 million SARS-CoV-2 infections and death rate statistics is accurate, and shows viral load? Why don't hundreds step forward and put their reputations on the line? If Flora Teoh has this wrong, then the stats used to propose lockdowns in the Third World are bogus and unreliable, yet those lockdowns are said to cause an extra 20 million deaths of innocent women and children from starvation, as well as ruining the world economy so much we can barely help the millions of others who would usually be in danger of starvation. Thus it is MEGA important these PCR Tests are accurate, but we get ONE scientist stepping forward to tell us they are. Why?
    Flora Teoh..... do you realise what you have done if you have this wrong???? MILLIONS OF CHILDREN AND BABIES may unnecessarily die in agony!
    In my opinion she uses a reprehensible and clearly false tactic. She accuses the writer of the "Manufactured Pandemic" article of attacking the brilliant science of Kary Mullis of PCR in general. That is nonsense. I do not believe that for one second. The person obviously just thinks the SARS-CoV-2 Test method of use is wrong, especially if Kaufman is right that they compare the RNA they extract to an equally corrupted SARS-CoV-1 RNA strand, and it's only 80% similar.

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    Kenneth McDonald3 months ago • edited
    If anyone tries to post the now famous article "Manufactured Pandemic" on Facebook, they are threatened by Facebook, and a link to Flora Teoh's article "PCR tests for COVID-19 are specific for the novel coronavirus SARS-CoV-2 and do not detect other coronaviruses, contrary to claims in viral article and video" is forced onto the post as a supposed refutation.
    Central to her claim the quick fix PCR test is accurate is her claim quote "Thanks to the efforts of scientists, we now know the full genome of SARS-CoV-2 [1]" and the link to "prove" this goes here. Can anyone here drag and drop a reply to me to show the "proof" of that from this article with a quote from it please?
    If they span out in a centrifuge the SARS-CoV-2 virus, and mapped its RNA. it helps prove the credibility of the PCR Tests for SARS-CoV-2 detection, but not viral load. But if they really did isolate the virus that way, why not then put it in a healthy person and cause the disease and fulfill either the Koch's or the River's postulates? That Dr Andrew Kaufman says have not been fulfilled, so therefore calling SARS-CoV-2 an infectious disease is outside of accepted science. So when did you fulfil the River's or Koch's postulates? (several questions there).

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    Rossana3 months ago
    The RdRp of RaTG13 has 100 % identity with the sequence BtCoV/4991
    (KP876546) identified by Ge and colleagues in a Rhinolophus affinis bat in the
    Yunnan province in 2013 as RaTG13. https://doi.org/10.1007/s12...
    BtCoV/4991 is a novel beta-CoV, clearly separated from all known alpha- and beta-CoVs at that time. BtCoV/4991 differentiates from other bat CoVs also in the phylogenetic analysis carried out by Wang and colleagues https://doi.org/10.3390/v11...
    Chen and colleagues identified BtCoV/4991 as the closest sequence to SARS-CoV-2 because RaTG13 had not yet been published at that time doi:
    10.1080/22221751.2020.1725399.
    How do BtCoV/4991 and RaTG13 relate to each other?

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    BatCoV5 months ago




    see more


    Sung-Joon Park5 months ago • edited
    We performed a metagenomic approach with WHU RNA-seq data;
    https://www.nature.com/arti...,
    (https://openlooper.hgc.jp/s...).
    Of note, WHU01 (SRR10903402) RNA-seq reads included statistically significant "Mycoplasma"-related reads, and the most probable species was "Mycoplasma hominis", while "Streptococcus"-related reads are probably not the human pathogen (contamination?) (https://openlooper.hgc.jp/f....
    Although this paper has used virus genomes, we have prepared the subsets of the RNA-seq reads that could not be mapped to the human genome and over 325,000 RefSeq microbial genomes (inc. SARS).
    Over 80% of the subset reads (R1 only) could be mapped to the nCoV genome (NC_045512), which may be greatly useful to characterize nCoV specific genome.

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    madmaxNY6 months ago
    Wow, unbelievable that this passed editorial and peer review at Nature! Something very strange that they would rush a paper out that is even not seriously edited, with tons of grammatical errors, so how many scientific errors either intentional or otherwise? Reviewers, please identify yourselves and release your reviews, the extent to which you went to validate the results, whether you had access to the raw sequence data, etc. And as cases continue to be made that the senior author on this paper, Zheng-Li Shi, has spent her career engineering this type of virus, publishing papers on the ability to do this and so on:
    https://www.zerohedge.com/g...
    I guess the editors at Nature did not feel it necessary to question the integrity of these results and force a more in depth review and replication from others.

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    bioconductor Sun6 months ago
    Where can the scientist world-wide get the raw NGS reads to review and re-analyze the squencing data?

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    Duhnkuhn Hussein6 months ago
    Zheng-Li Shi will end up killing more people that Mao Zedong. That is impressive.


    Jason6 months ago • edited
    some key data and logical connection is missing in order for the author to claim "We then found a short RdRp region from a bat coronavirus termed BatCoV RaTG13 which we previously detected in Rhinolophus affinis from Yunnan Province showed high sequence identity to nCoV-2019."


    BiologyGeek6 months ago • edited
    Tens of thousands of people are infected and hundreds died. We need to better understand how the bat coronavirus got transmitted to human patients in this 2020 outbreak. Many blame a wildlife market in Wuhan - but there are 661 cities in China and many of them, especially 41 in Guangdong where residents are known to like bushmeat, have such markets. Why didn't the outbreak start from any of the other 660 cities, especially the 41 in Guangdong - but why Wuhan? For example, the 2002-2003 SARS outbreak started from Fushan, Guangdong, but not Wuhan.
    Moreover, according to Figure 1 of China CDC's recent report: https://www.nejm.org/doi/fu..., the first two patients had no link to the Wuhan wildlife market at all.
    Now, if you look at this: there are 661 cities in China, but there is only one city, Wuhan, hosting a biosafety level 4 (BSL-4, or P4) lab. It's the only lab in China that captures bats and is authorized to study the bat coronavirus. The authors, as part of this BSL-4 lab, started to do so since 2015. Statistically, the bat coronavirus of the Wuhan outbreak is more likely to be from the only Wuhan BSL-4 lab that studies bat coronavirus in China, as opposed to be from the wildlife markets - Wuhan's wildlife market is among hundreds of wildlife markets existing in hundreds of cities in China. Is there any possibility that the bat coronavirus got leaked from this BSL-4 lab unintentionally through improper handling of samples and lab animals? This needs to be investigated.
    • 6

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    • BiologyGeek BiologyGeek6 months ago • edited
      An update on the questions on the origin of the coronavirus:
      A recent news reported that China has denied the lab link to coronavirus. https://www.washingtontimes... "However, a Chinese government-funded study published Jan. 24 in the medical journal The Lancet by 29 Chinese scientists found that 13 of 41 victims surveyed had no connection to the wild animal market. Significantly, the first patient identified with the coronavirus was a man who reported pneumonia-like symptoms on Dec. 1 but had no connection to the market."
      The mentioned Lancet paper is here: https://www.thelancet.com/j...
      Who is the first patient? What happened to him? How come he had no link to the market? Why the coronavirus he contracted with "showed high sequence identity" to the "BatCoV RaTG13" stored in the lab of the authors?


      BiologyGeek BiologyGeek6 months ago • edited
      Also, we need to note that, as recently publicly pointed out by Dr. Yi Rao, President of Capital University of Medical University in China, Mrs. Yan-yi Wang - the Lab Director of the Wuhan BSL-4 lab as well as the supervisor of Dr. Zheng-li Shi - is professionally not qualified. News is here: https://www.backchina.com/n....
      Moreover, the concern about the safety issue of the Wuhan BSL-4 lab was discussed as early as in 2017: https://www.nature.com/news...
      The authors of this article are from the questioned Wuhan BSL-4 lab. The safety concern is serious. The possibility of an unintentional bat coronavirus leak causing this 2020 coronavirus outbreak should be at least discussed and investigated.


    Michael Siedel6 months ago
    just a few questions? 22 pages seven test persons only one had the virus detectable! all slight fever! 96 percent match! the rest of the other ingredients thrown together wildly? who cross-checks which laboratory level 4? why 9 days until approval by Nature, since submission when it is urgent? and then another 4 to upload 22 pages? Thank you very much! yes always the bats and always the markets and always test environment Wuhan! Dankeschön ihr Hausmeister Krause/ #fafnör
    Michael Siedel member MinD Germany number 21165


    Parvaiz 6 months ago
    Nitazoxanide as a possible agent for the 2019-Novel Corona Virus infection
    Isolation of the virus and testing of antiviral drugs is critical to the management of the cases at present as this outbreak seems to have shaped clearly worse than the SARS and the MERS-coV outbreaks reported in the earlier parts of this century. This would allow studying the antiviral efficacy of various antiviral agents against the virus. Pertinently combinations of antiviral agents can be tried. Additionally the antiparasitic agent nitazoxanide might be useful and may be worthwhile testing in patients who are sick based on the premise that the combination of nitazoxanide and oseltamivir was found to be superior to oseltamivir alone in influenza patients in a previous study. Nitazoxanide is a safe antiparasitic agent, widely prescribed and has been demonstrated to have antiviral properties which could be exploited in the current settings to treat severe cases in combination with investigational antiviral agents as also test its efficacy in the laboratory against cultured virus isolates.
This is leading me to look further into other articles, and other papers eventually too, two I will share in this post, one is from an interview with one of the main authors of the above paper (link from the most recent comment posted Acron Naji)
“Correspondence to Zheng-Li Shi.”


Reply to Science Magazine

https://www.sciencemag.org/sites/default/files/Shi Zhengli Q&A.pdf
(page 7)
(7)What about the cave at Mojiang in 2013? When did you first isolate RaTG13? When did you complete the full sequencing of it?A:We detected the virus by pan-coronavirus RT-PCR in a bat fecal sample collected from Tongguan town, Mojiangcounty in Yunnan provincein 2013, and obtained its partial RdRp sequence. Because the low similarity of this virus to SARS-CoV, we did not pay special attention to this sequence. In 2018, as the NGS sequencing technology and capability in our lab was improved, we did further sequencing of the virus using our remaining samples, and obtained the full-length genome sequence of RaTG13 except the 15 nucleotides at the 5’ end. As the sample was used many times for the purpose of viral nucleic acid extraction, there was no more sample after we finished genome sequencing, and we did not do virus isolation and other studies on it. Among all the bat samples we collected, the RaTG13 virus was detected in only one single sample. In 2020, we compared the sequence of SARS-CoV-2 and our unpublished bat coronavirus sequences and found it shared a 96.2%identity with RaTG13. RaTG13 has never been isolated or cultured.
So Zheng-Li Shi in this interview has admitted that in her earlier work 'discovering' the RaTG13 virus, it was never “isolated” and so was also not grown in culture nor electron micrograph and virus particle analysis etc. nor any “koch” postulate disease cell culturing work etc. etc. I think this may well have strong relevance to the work in this paper here alleging to have “isolated” 'sars-cov-2' also, but I need to fully break down this article (and at least look at the many extra data tables/sets etc.) and see how vigorous or valid is the alleged “isolation”, that is a real bitch of a task, so this will be a several part series of posts on this paper/article.
The other is from one of the commenter's who wrote a short critique of the this article, a brief look now,

1Short note
Understanding the origin of‘BatCoVRaTG13’, a virus closest to SARS-CoV-2 Monali C. Rahalkar1 and Rahul A. Bahulikar2
(not peer-reviewed of course yada yada)
https://www.preprints.org/manuscript/202005.0322/v2
Abstract

Genomic analysis indicates that SARS-CoV-2 is most related to RaTG13, a beta coronavirus derived from bats by 96% 1. At present, RaTG13 is only available on the public database in the form of a genome sequence. The genome of RaTG13 (MN996532.1) was sequenced from the RNA of a bat faecal swab collected in 2013 from Yunnan, China, however the exact location is not mentioned. Since RaTG13 is one of the main supports for SARS-CoV-2 to have a natural origin, it is of utmost importance to understand the sample location. RNA dependent RNA polymerase (RdRp) sequence of RaTG13 shows that it is 100% similar to that of bat corona virus BtCoV/4991 and 98.7-98.9% similar to SARS-CoV-2 RdRp2. BtCoV/4991 was described to be a SARS-like (SL-) corona virus from bat faeces sampled in an abandoned mine from Mojiang2. Both the publications1,2 are authored by Dr. Zheng-li Shi(Z-L Shi),who is described as the bat woman of China3.However, BtCoV/4991 has not been mentioned by Zhou et al 2020 1 where novel corona virus was first described. Based on the RdRp sequence similarities, similarities in sample collection dates, sample locations, and the fact thatRaTG13 is mentioned synonymous to BtCoV/4991on the Chinese bat database, it is predicted that RaTG13 and BtCoV/4991originate from the same sample. The sample, bat faecal swab was collected in 2013 from an abandoned mineshaft in Mojiang by Dr. Shi and her work group. In 2012, in a Mojiang mineshaft, six mine workers suffered from atypical pneumonia and three of them died. These workers were engaged in the work of clearing debris from a mineshaft which had a lot of bats and bat faeces3,4.A detailed health investigation indicated that the miners suffered from atypical pneumonia mostly of the viral origin4. Therefore,in the light of the present Covid-19 caused by SARS-CoV-2, the fact that its phylogenetic neighbour RaTG13 originated from bat faeces collected from a mineshaft, which was also the origin of pneumonia-like disease in miners in 2012,should be noted.
I will note that Dr. Zheng-li Shi in Science Magazine interview stated on a later page that the bat coronavirus BtCoV/4991 was renamed as RaTG13 so they are the exact same “virus' according to her; and I assume that the Science Mag interview came out after Monali C. Rahalkar's and Rahul A. Bahulikar's critique in which I think they say that the virus identified as bat coronavirus RaTG13 genomic sequence was not reported/published until after the paper for '2019-nCoV' was published.
 

gl69m

Member
Continuing on from original thread, post #30 (from gl69m)
Can't do a follow up to post #29 yet, I saw this recently, CDC pcr protocol document has been updated:

last two weeks, Facebook commenting exchange (I posted this pcr protocol update, like the sheople morons there cared of course not) that lost me a non-friend on my FB friend list over 'covid' being a hoax and they believe this non-sense and refused or couldn't/wouldn't show evidence for people they know that supposedly died of 'covid' or was recently diagnosed, for my rants I was de-friended, which I'm glad about that- woman could care fucking less about the economic cost to people out there in the U.S. or anywhere in the world suffering from the shutdowns, her bullshit logic was if the insane measures keep her mother from getting 'covid' than fuck everyone else's "economy" and to that I say fuck people like her and all 'curve flatteners' and mask shamers, their actions supporting this insanity will kill far more people in the long run than a real pandemic (economic and food/resource deprivation) and they know this and still don't give a fuck and fuck the so-called 'covid' deaths they are not more sanctimonious deaths/losses (even if we believed this 'pandemic' was real) than lives lost to any other cause of death and certainly not least death from starvation- fuck those selfish motherfucking people!!! Ok end rant, no to the post.

from post #4, built in bias (for generating false positives, bias #17) for the CDC pcr protocol for 'covid-19',

Quote:

Summary of built in biases (that strongly favor a “positive' result outcome{which is most likely to be an actual “false positive” IMO} for 2019-nCoV) within this protocol:

DC-006-00019, Revision: 03 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 3/30/2020CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel For Emergency Use OnlyInstructions for UseCatalog # 2019-nCoVEUA-01 1000 reactionsFor In-vitro Diagnostic (IVD) UseRx Only
https://www.fda.gov/media/134922/download

17- no quantified virus isolates of the 2019-nCoV are currently available (page 38 ),assays designed for detection of the 2019-nCoV RNA were tested with characterized stocks of in vitro transcribed full length RNA. Is this a possible admission the virus is fake and that the '2019-nCoV' genome may be a mock (theoretical genome)? Well, I think it's possible: my tinfoil hat dunning-kreuger senses (not spidy senses
biggrin.gif
) are tingling, how about yours?

from post #5

Quote:

17- From page 38,
Quote:
Performance Characteristics
Analytical Performance:

Limit of Detection (LoD):

LoD studies determine the lowest detectable concentration of 2019-nCoV at which approximately 95% of all (true positive) replicates test positive. The LoD was determined by limiting dilution studies using characterized samples.

The analytical sensitivity of the rRT-PCR assays contained in the CDC 2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel were determined in Limit of Detection studies. Since no quantified virus isolates of the 2019-nCoV are currently available, assays designed for detection of the 2019-nCoV RNA were tested with characterized stocks of in vitro transcribed full length RNA (N gene; GenBank accession: MN908947.2) of known titer (RNA copies/μL) spiked into a diluent consisting of a suspension of human A549 cells and viral transport medium (VTM) to mimic clinical specimen.
I'm thinking that normally there is quantified virus isolates to validate test methods normally, here they did their fast-tracked validation with “transcribed” full length RNA of the N gene, from the genome published from this GenBank accession deposit MN908947.2. This is perhaps the second genome version of the original genome published by Chinese scientists (I think on Jan 5th 2020) for the alleged 2019-nCoV virus, subsequently renamed SARS-CoV2 virus.

Is it possible that no quantified virus isolates of the 2019-nCoV were currently available, because they didn't (don't exist)? Is this genome perhaps a mock genome, and this transcribed RNA put into this test validation (and for the 2019-nCoV positive controls also in subsequent actual specimen testing?) was actually artificially printed nucleic acid? I have a hunch (
icon_think.gif
).

Update to this document from above link (same link as in post #4, https://www.fda.gov/media/134922/download, this time on page 39):

Quote:

CDC-006-00019, Revision: 04 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 6/12/2020

CDC 2019-Novel Coronavirus (2019-nCoV)
Real-Time RT-PCR Diagnostic Panel
For Emergency Use Only
Instructions for Use
Catalog # 2019-nCoV
EUA-01 1000 reactions
For In-vitro Diagnostic (IVD) Use
Rx Only

Page 39

Performance Characteristics


Analytical Performance:

Limit of Detection (LoD):

LoD studies determine the lowest detectable concentration of 2019-nCoV at which approximately 95% of all (true positive) replicates test positive. The LoD was determined by limiting dilution studies using characterized samples.

The analytical sensitivity of the rRT-PCR assays contained in the CDC 2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel were determined in Limit of Detection studies. Since no quantified virus isolates of the 2019-nCoV are currently available, assays designed for detection of the 2019-nCoV RNA were tested with characterized stocks of in vitro transcribed full length RNA (N gene; GenBank accession: MN908947.2) of known titer (RNA copies/μL) spiked into a diluent consisting of a suspension of human A549 cells and viral transport medium (VTM) to mimic clinical specimen. Samples were extracted using the QIAGEN EZ1 Advanced XL instrument and EZ1 DSP Virus Kit (Cat# 62724) and manually with the QIAGEN DSP Viral RNA Mini Kit (Cat# 61904). Real-Time RT-PCR assays were performed using the ThemoFisher Scientific TaqPath™ 1-Step RT-qPCR Master Mix, CG (Cat# A15299) on the Applied Biosystems™ 7500 Fast Dx Real-Time PCR Instrument according to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel instructions for use.

So the update on June 12th, I haven't looked at much of the rest of the document yet: and this time this section is on page 39 instead of page 38 so there has to be some change somewhere: however this wording looks basically the same, and so they don't appear to have added new testing or further validation of the method that I can see skimming some of the other pages; but the big piece the gargantuan pink elephant in the room is that they still don't mention using any "quantified" (and therefore not "isolated") virus stocks or in other words didn't use isolated/quantified 'sars-cov2' virus, why the fuck not??!! surely there has been plenty of time for them to generate these viral stocks, if this virus were real?? Seriously. To me this kind of feels like another smoking gun, I may have to include that on the other 'covid' smoking guns thread too.

Post #31 (from original thread, from gl69m)
New revision (05) for the CDC 'covid' pcr protocol document:

https://www.fda.gov/media/134922/download

Quote:


CDC 2019-Novel Coronavirus (2019-nCoV)

Real-Time RT-PCR Diagnostic Panel

For Emergency Use Only
Instructions for Use
Catalog # 2019-nCoVEUA-01

1000 reactions
For In-vitro Diagnostic (IVD) Use
Rx Only

CDC-006-00019, Revision: 05 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 07/13/2020


from page 39


Performance Characteristics

Analytical Performance:

Limit of Detection (LoD):
LoD studies determine the lowest detectable concentration of 2019-nCoV at which approximately 95% of all (true positive) replicates test positive. The LoD was determined by limiting dilution studies using characterized samples.

The analytical sensitivity of the rRT-PCR assays contained in the CDC 2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel were determined in Limit of Detection studies. Since no quantified virus isolates of the 2019-nCoV are currently available, assays designed for detection of the 2019-nCoV RNA were tested with characterized stocks of in vitro transcribed full length RNA (N gene; GenBank accession: MN908947.2) of known titer (RNA copies/μL) spiked into a diluent consisting of a suspension of human A549 cells and viral transport medium (VTM) to mimic clinical specimen. Samples were extracted using the QIAGEN EZ1 Advanced XL instrument and EZ1 DSP Virus Kit (Cat# 62724) and manually with the QIAGEN DSP Viral RNA Mini Kit (Cat# 61904). Real-Time RT-PCR assays were performed using the ThemoFisher Scientific TaqPath™ 1-Step RT-qPCR Master Mix, CG (Cat# A15299) on the Applied Biosystems™ 7500 Fast Dx Real-Time PCR Instrument according to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel instructions for use.

Wording here is the same as revision 04 from a month ago (6/12/20).

Here is the revision history from page 50,

Quote:

Revision HistoryRevision#Effective DateSummary of Revisions1February 4, 2020Original Instructions for Use2March 15, 2020•Intended use update•Removal of N3 primer and probe set from Diagnostic Panel•Performance data update•Addition of alternative nucleic acid extraction platforms•Addition of acceptable alternatives to HSC and addition of QIAGEN RUO extraction reagents•Positive results no longer presumptive. No confirmation of positive results required3March 30, 2020•Addition of alternative enzyme master mix options4June 12, 2020•Addition of MagNA Pure 24 extraction method•Addition of performance data for the MagNA Pure 96 extraction method with SARS-CoV-2 •Addition of heat treatment alternative to specimen extraction •Addition of Roche and QIAGEN external lysis buffer alternatives•Acknowledgment of FDA policy permitting end users to qualify alternative components without seeking an EUA or EUA amendment5July 13, 2020•Addition of Promega Maxwell® RSC 48 extraction method•Updateto in silicoinclusivity analyses

Not going to clean that up or screen capture that for now, I may look at it later, some additions are protocols for added approved product lines etc. The two that are peaking my interest is from revision 04

"•Addition of heat treatment alternative to specimen extraction"

and

"•Acknowledgment of FDA policy permitting end users to qualify alternative components without seeking an EUA or EUA amendment

Have to check those out later.

Post #32 (from original thread, from gl69m)
Need to break this article down, hopefully soon, it contains alleged evidence for 'isolation' of 'sars-cov2', for me it isn't very convincing; I will at least make a relatively detailed argument for it not being really "isolated' here, but that takes considerable time for me to do that thoroughly.

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

Published: 03 February 2020

https://www.nature.com/articles/s41586-020-2012-7

Post #33 (from original thread, from nrms)
Just a quick mention here to Kary B Mullis. I didnt realize he had died so recently.
Kary Banks Mullis (December 28, 1944 – August 7, 2019)
That is just two months before Event 201.

Good luck with that gl69m. Other doctors that are looking for 'proof of isolation' it are not finding it.

Post #34 (from original thread, from gl69m)
from CDC website about Antibody tests:

https://www.cdc.gov/coronavirus/2019-ncov/testing/serology-overview.html
Test for Past Infection (Antibody Test)

If you test positive or negative for COVID-19 on a viral or an antibody test, you still should take preventive measures to protect yourself and others.

We do not know yet if people who recover from COVID-19 can get infected again. Scientists are working to understand this.

What do your results mean?

If you test positive

  • A positive test result shows you may have antibodies from an infection with the virus that causes COVID-19. However, there is a chance a positive result means that you have antibodies from an infection with a virus from the same family of viruses (called coronaviruses), such as the one that causes the common cold.
  • Having antibodies to the virus that causes COVID-19 may provide protection from getting infected with the virus again. If it does, we do not know how much protection the antibodies may provide or how long this protection may last.
  • Talk with your healthcare provider about your test result and the type of test you took to understand what your result means. Your provider may suggest you take a second type of antibody test to see if the first test was accurate.
  • You should continue to protect yourself and others since you could get infected with the virus again.
    • If you work in a job where you wear personal protective equipment (PPE), continue wearing PPE.
  • You may test positive for antibodies even if you have never had symptoms of COVID-19. This can happen if you had an infection without symptoms, which is called an asymptomatic infection.
If you test negative
  • You may not have ever had COVID-19. Talk with your healthcare provider about your test result and the type of test you took to understand what your result means.
  • You could still have a current infection.
    • The test may be negative because it typically takes 1–3 weeks after infection for your body to make antibodies. It’s possible you could still get sick if you have been exposed to the virus recently. This means you could still spread the virus.
    • Some people may take even longer to develop antibodies, and some people who are infected may not ever develop antibodies.
If you get symptoms after the antibody test, you might need another test called a viral test.

Regardless of whether you test positive or negative, the results do not confirm whether or not you are able to spread the virus that causes COVID-19. Until we know more, continue to take steps to protect yourself and others.

Learn more about using antibody tests to look for past infection.
I wanna repeat this for those in the back who can't see or hear that well:
  • A positive test result shows you may have antibodies from an infection with the virus that causes COVID-19. However, there is a chance a positive result means that you have antibodies from an infection with a virus from the same family of viruses (called coronaviruses), such as the one that causes the common cold.
(there is a shitload more than just "a chance" that any antibodies they "detect' were produced from something other than 'sars-cov2', IMSO)

Two other points worth repeating,
  • You may test positive for antibodies even if you have never had symptoms of COVID-19. This can happen if you had an infection without symptoms, which is called an asymptomatic infection.
  • Some people may take even longer to develop antibodies, and some people who are infected may not ever develop antibodies.
    Everybody confused as hell yet? Good!

    Even more confusion,

    GUIDANCE ON
    INTERPRETING COVID-19 TEST RESULTS

    https://www.whitehouse.gov/wp-content/uploads/2020/05/Testing-Guidance.pdf

DO NOT Get Tested or YOUR Life Will Never Be Your Own Again
Jul 15, 2020

https://www.youtube.com/watch?v=qdgsL... Roy is a former US Army LTC of 28 years. He served in various Military Police and Military Intelligence positions around the globe. He was also a municipal police officer for about 3 years. All credit to: roypotterqa
...

Roy Potter I'm guessing, one of Harry's uncles; he suggests that the tests are delivering the experimental vaccine(s). I've heard also that they may be delivering other infections etc. WHO knows for sure, I don't, but I sure as hell don't want any of these 'covid' tests whatsoever if I can avoid for sure.
 

gl69m

Member
Post #36 (from original thread, from gl69m)

Coming back to Post #35 (as a part 2 here), starting off with this point in the article,

Published 03 February 2020
A pneumonia outbreak associated with a new coronavirus of probable bat origin
https://www.nature.com/articles/s41586-020-2012-7
The close phylogenetic relationship to RaTG13 provides evidence that 2019-nCoV may have originated in bats.

As we saw in Post #35 that the main author Zheng-Li Shi admitted that this virus RaTG13 was never actually “isolated” in the original work done on this virus, and so that should at least on it's face call into question the alleged sequencing of said RaTG13 genome. And so if we can call into question this genome does it have any relevance towards the validity or calling into question the validity of 'sars-cov2'? I think it does, and I realize also that without further investigation of this article and other information I can't fully defend that position or opinion and be seen as “objective” as it were. So let's get into the more of this paper, following the last section quoted,
We rapidly developed a qPCR-based detection method on the basis of the sequence of the receptor-binding domain of the S gene, which was the most variable region of the genome (Fig. 1c). Our data show that the primers could differentiate 2019-nCoV from all other human coronaviruses including bat SARSr-CoV WIV1, which shares 95% identity with SARS-CoV (Extended Data Fig. 4a, b). Of the samples obtained from the seven patients, we found that six BALF and five oral swab samples were positive for 2019-nCoV during the first sampling, as assessed by qPCR and conventional PCR. However, we could no longer detect virus-positive samples in oral swabs, anal swabs and blood samples taken from these patients during the second sampling (Fig. 2a). However, we recommend that other qPCR targets, including the RdRp or envelope (E) genes are used for the routine detection of 2019-nCoV. On the basis of these findings, we propose that the disease could be transmitted by airborne transmission, although we cannot rule out other possible routes of transmission, as further investigation, including more patients, is required.
1637212294105.png1637212470417.pnga, Molecular detection of 2019-nCoV in seven patients. Patient information can be found in Extended Data Tables 1, 2. Detection methods are described in the Methods. AS, anal swab; OS, oral swab. b, Dynamics of 2019-nCoV antibody levels in one patient who showed signs of disease on 23 December 2019 (ICU-06). OD ratio, optical density at 450–630 nm. The right and left y axes indicate ELISA OD ratios for IgM and IgG, respectively. c, Serological test of 2019-nCoV antibodies in five patients (Extended Data Table 2). The asterisk indicates data collected from patient ICU-06 on 10 January 2020. b, c, The cut-off was to 0.2 for the IgM analysis and to 0.3 for the IgG analysis, according to the levels of healthy controls.

One interesting point here is that the main sample that '2019-nCoV' ('sars-cov2') was “sequenced' (and the alleged new 'virus' 'isolated' from) is that in Figure 2a this sample WIV04 (is also ICU-06 in other data tables and graphs) was either 0 for log copies on the PCR for the genetic copies of alleged '2019-nCoV' on the OS (oral swab) or no oral swab was performed on this patient only a BALF. And that upon sampling for all patients on Jan 10th 2020 there was no PCR copies detected in oral swab or anal swab or blood. Blood or anal swab was apparently not tested for the Dec 30th 2019 sampling, and no BALFs for the January 10th 2020 sampling.

Our data show that the primers could differentiate 2019-nCoV from all other human coronaviruses including bat SARSr-CoV WIV1, which shares 95% identity with SARS-CoV (Extended Data Fig. 4a, b).”

These primers apparently based on just the receptor binding domain of the S (spike) protein in which '2019-nCoV' is 93.6% identical with RaTG13. Because of the lower homology of sequence of other CoV's compared the sequence generated for '2019-nCoV' it should be expected primers targeting the S protein RBD region ought to be distinguishable, but so far no 'virus” had been “isolated” to take and purify then isolate and replicate in culture and make sure you have only one virus and then break all the protein and RNA down to sequence it knowing you're sequencing an actual virus isolated from a patient/specimen sample, but here the sequencing (and “serology” detection as well) precedes the alleged viral 'isolation', seeming a bit backwards it might seem especially if you think it's a new virus, or that's my opinion of it but maybe most virologists would say that's incorrect but I don't know that at this time. Moving on.

Is it possible that the coronavirus genetic material 'detected” from the panel of the pan-CoV primers simply created an amalgamation of a 'genome' stitiched together here from multiple viruses present, and some of them not even coronaviruses? I think that's possible of course.
Next step they report is “serological' detection:

For serological detection of 2019-nCoV, we used a previously developed nucleocapsid (N) protein from bat SARSr-CoV Rp3 as antigen for IgG and IgM enzyme-linked immunosorbent assays (ELISAs), as this protein shared 92% amino acid identity to N protein of 2019-nCoV (Extended Data Fig. 5) and showed no cross-reactivity against other human coronaviruses except SARSr-CoV7. We were only able to obtain five serum samples from the seven patients with viral infections. We monitored viral antibody levels in one patient (ICU-06) 7, 8, 9 and 18 days after the onset of disease (Extended Data Table 2). A clear trend was observed in the IgG and IgM titres, which increased over time, except that the IgM titre was decreased in the last sample (Fig. 2b). As a second analysis, we tested samples from 5 of the 7 virus-positive patients around 20 days after disease onset for the presence of viral antibodies (Extended Data Tables 1, 2). All patient samples—but not samples from healthy individuals—were strongly positive for viral IgG (Fig. 2b). There were also three IgM-positive samples, indicating an acute infection.

So the N protein (Nucleocapsid) from a generic bat coronavirus (bat SARSr-CoV Rp3) was used as an antigen to 'detect” IgM and IgG anti-bodies in the patient serum samples. The N protein is generally known to be at least 90% (nucleotide and sometimes more conserved amino acid sequence) conserved sequence in most coronaviruses, so relatively high potential for cross reactivity of antibodies to certain antigens from multiple different viruses. But they say this antigen “and showed no cross-reactivity against other human coronaviruses except SARSr-CoV7.”, except SARSr-CoV but that seems to be a generic set of coronaviruses (bat and human?) and not just one "coronavirus" so that seems quite ambiguous or confusing to me,

The article referenced here, SARSr-CoV7.(reference 7), I have looked at this article in at least one other post somewhere (not sure where at the moment), and I noted there was some cross reactivity to at least three other human coronaviruses, OC43 and HKU1 and NL-63,

Serological Evidence of Bat SARS-Related Coronavirus Infection in Humans, China
2018 Feb Published online 2018 Mar 2
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178078/
Polyclonal antibodies against each of the six NPs were prepared in rabbits as previously published (He et al. 2006). Cross-activity was evaluated with ELISA and Western blot (Supplementary Figures S1, S2). No significant cross-activity was detected among NPs and their corresponding antibodies for Rp3, MERS-CoV, NL63, or 229E. Cross-reaction was detected between OC43 and HKU1 as reported previously (Lehmann et al. 2008).
The degree of reactivity in Western blot correlated well with the ELISA OD readings, providing further confidence in the ELISA screening method. None of the sera reacted with NPs of either MERS-CoV or EBOV. On the other hand, all 10 human sera (9 from Jinning and 1 from Wuhan), regardless of their Rp3 NP reactivity, reacted strongly with the NL63 NP as expected due to high prevalence of NL63 infection in humans worldwide (Abdul-Rasool and Fielding 2010).

Incidentally Zheng-Li Shi is a principle author of all three papers here, one for RaTG13 in 2013 and this bat related SARS paper from 2018 and now in 2020 the defining paper for 'sars-cov2' “discovery/sequencing' and 'isolation'. She is also from the WIV (Wuhan Institute of Virology) possibly the same group given funding by Fraudci/NIH (and WHO?) in 2014 to study possible gain of function experiments on coronaviruses (ACEII receptors), I'll have to check on that. According to fact check from USA Today,

Fact check: Obama administration did not send $3.7 million to Wuhan lab
May 4, 2020
https://www.usatoday.com/story/news...tion-did-not-send-3-7-m-wuhan-lab/3061490001/
EcoHealth Alliance and coronavirus research

The EcoHealth Alliance is a nongovernmental research group that focuses on emerging diseases caused by human and animal interactions. The National Institutes of Health has consistently funded the organization for projects since at least 2002.
(so the NIH has funded this group EcoHealth Alliance since 2002, didn't start with Obama,)
In 2014, the NIH approved a grant to EcoHealth Alliance designated for research into “Understanding the Risk of Bat Coronavirus Emergence.” The project involved collaborating with researchers at the Wuhan Institute of Virology to study coronaviruses in bats and the risk of potential transfer to humans.
(but this project did start with the Obama administration,)

The original five-year grant was reapproved by the Trump administration in July 2019. In total, $3,378,896 in NIH funding was directed from the government to the project. (and then the project was continued by the Trump administration, not that long before Event 201 in Oct 2019)
The project, which was established “to understand what factors allow coronaviruses, including close relatives to SARS, to evolve and jump into the human population,” yielded 20 scientific reports on how zoonotic diseases may transfer from bats to humans.

Over the course of the two grants approved by the NIH for EcoHealth Alliance, the Wuhan Institute received about $600,000 from the NIH, according to Robert Kessler, a spokesperson for EcoHealth Alliance. The funding was a fee for the collection and analysis of viral samples.


“It's hard to do this work in other countries. Very complicated. It requires a lot of traveling. It would be so convenient if we could do it in our own backyards,” Peter Daszak, president of the EcoHealth Alliance, ( Peter Daszak was named as an included author in two of these three papers of CoV's {and 'sars-cov2'} whom the main author is Zheng-Li Shi) told USA TODAY. “The viruses that are a high risk to public health are not in the U.S., they are in China. If we want to know anything about the next pandemic, we need to be working in the countries where these viruses are.”

The Wuhan Institute of Virology is a state-controlled research institute in Wuhan, China, the city where SARS-CoV-2 probably originated. Established in 2015, the laboratory was China’s first Level 4 biosafety facility, the highest standard meant to handle highly infectious or otherwise deadly pathogens.

Our ruling: Partly false

We rate this claim PARTLY FALSE because some of it was not supported by our research. It is misleading to claim that the Obama administration gave funding to a Chinese research institute. It is true that funds were provided to a project where an American research group worked alongside a Chinese organization. Claims that the funding helped produce the pandemic are unsubstantiated. A total of $3.7 million was not given to the Wuhan Institute of Virology, only about $600,000.

So next they claim to have performed the viral 'isolation” of '2019-nCoV',
We next successfully isolated the virus (called 2019-nCoV BetaCoV/Wuhan/WIV04/2019) from both Vero E6 and Huh7 cells using the BALF sample of patient ICU-06. Clear cytopathogenic effects were observed in cells after incubation for three days (Extended Data Fig. 6a, b). The identity of the strain WIV04 was verified in Vero E6 cells by immunofluorescence microscopy using the cross-reactive viral N antibody (Extended Data Fig. 6c, d) and by metagenomics sequencing, most of the reads of which mapped to 2019-nCoV, and qPCR analysis showed that the viral load increased from day 1 to day 3 (Extended Data Fig. 6e, f). Viral particles in ultrathin sections of infected cells displayed a typical coronavirus morphology, as visualized by electron microscopy (Extended Data Fig. 6g). To further confirm the neutralization activity of the viral IgG-positive samples, we conducted serum-neutralization assays in Vero E6 cells using the five patient sera that were IgG-positive. We demonstrate that all samples were able to neutralize 100 TCID50 (50% tissue-culture-infective dose) of 2019-nCoV at a dilution of 1:40–1:80. We also show that this virus could be cross-neutralized by horse anti-SARS-CoV serum (gift from L.-F. Wang) at dilutions of 1:40; however, the potential for cross-reactivity with SARS-CoV antibodies needs to be confirmed with anti-SARS-CoV serum from humans (Extended Data Table 4).

Here they claim to have used Vero E6 cells to culture the virus from patient samples and use that culture for electron micrographs and supposedly neutralized the virus with serum samples from the patients: which very interestingly this 'virus” could be neutralized with serum from horse (inoculated with SARS-CoV (so confirms very likely serolgical antibody cross-reactivity it would seem to me produced from the original SARS-CoV virus) and they say cross reactivity needs to be confirmed from human serum exposed to SARS-CoV. I have seen at least a few papers claiming to show no cross-reactivity with SARS-CoV antibodies, not looking at that now, and they didn't look all that convincing to me but that's beyond my scope at present here. I still have to wonder why that would be much of a concern or issue if the SARS-CoV virus was allegedly no longer circulating in any populations worldwide since supposedly 2004 particularly in China; well except that it is claimed that polyclonal antibodies or antigens from SARS-CoV can thus be used for "serological" detection of possible 'sars-cov-2' present or past infection (asymptomatic or not).

Another paper I saw states that BALF samples from patients in Wuhan were cultured on human airway epithelial cells (for viral “isolation') and not Vero E6 cells (or Huh7 cells) as claimed in this paper, has a different set of authors it looks like without Zheng-Li Shi credited to this paper just below (I think perhaps this paper is credited to a Chinese CDC team of investigators), where it is claimed also that de novo sequencing was done from BALF samples and from the 'viral' isolates from the human airway epithelial cell cultures whereas I don't think in Dr. Shi's '2019-nCoV' paper that de novo sequencing was done from the actual 'viral' isolates (from VeroE6 and Huh7 cells).

A Novel Coronavirus from Patients with Pneumonia in China, 2019
N Engl J Med. 2020 Feb 20;
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092803/
however three of the samples used look like possibly are three of the same patients used in this study,

To further characterize the virus, de novo sequences of 2019-nCoV genome from clinical specimens (bronchoalveolar-lavage fluid) and human airway epithelial cell virus isolates were obtained by Illumina and nanopore sequencing. The novel coronavirus was identified from all three patients. Two nearly full-length coronavirus sequences were obtained from bronchoalveolar-lavage fluid (BetaCoV/Wuhan/IVDC-HB-04/2020, BetaCoV/Wuhan/IVDC-HB-05/2020|EPI_ISL_402121), and one full-length sequence was obtained from a virus isolated from a patient (BetaCoV/Wuhan/IVDC-HB-01/2020|EPI_ISL_402119).

(are these three patients the same as 2019-nCoV BetaCoV/Wuhan/WIV04/2019, 2019-nCoV BetaCoV/Wuhan/WIV05/2019 and 2019-nCoV BetaCoV/Wuhan/WIV01/2019? It seems quite possible to me, according that the phylogenetic tree presented in both papers look very similar, except that RaTG13 is not included in this paper: and further the difference is that de novo sequencing {to get precise genome} is said to have been done in this paper using the viral 'isolates” and in Zheng-Li Shi's paper the de novo sequencing was done on the BALF samples before the 'isolation'.

So I want to pause again for further information regarding two other critiques of the author Zheng-Li Shi and this paper alleging 'discovery' and 'isolation' of '2019-nCoV' aka 'sars-cov-2'.

Was the coronavirus created by Chinese scientist who tried to cover her tracks — and failed?
Dr. Shi Zhengli's January 2020 'discovery' was the equivalent of trying to wipe the fingerprints from a gun that had just been used to commit murder
Steven W. Mosher
May 19, 2020
https://www.pop.org/was-the-coronav...ist-who-tried-to-cover-her-tracks-and-failed/
Dr. Shi and her superiors, undoubtedly including Major General Chen Wei, the head of the PLA’s bioweapons program, knew that the coronavirus she had assembled using recombinant technology was so different from other known coronaviruses that it would raise suspicions. None of the other known beta-coronaviruses, the family from which her “backbone” coronavirus came from, had anything resembling the genetic sequence she had inserted to make it more infectious to humans.

To reinforce the “Wet Market” cover story, namely, that this new pathogen had come from nature and not from her lab, something had to be done. And it had to be done quickly, since by then the China Coronavirus had spread to the rest of the world. Anger against China for its lack of transparency about the origins and characteristics of the virus was growing.

Dr. Shi decided to “discover” a new bat coronavirus that was very similar to the one she had created. That “discovery” would prove that coronaviruses similar to SARS-CoV-2 were found in nature, and so deflect the growing suspicion that she had engineered it in her lab.

The similarity between the two coronaviruses—including their common ability to infect humans–would greatly reinforce her story that the SARS-CoV-2 had jumped from a bat to a human, perhaps through some intermediate species at the Wuhan wet market.

So, all Dr. Shi had to do was sit down before her computer keyboard, open a word file, and begin to fabricate the SARS-CoV-2 analogue that she would claim to have found in nature seven years before. All she had to do was type in the genetic sequence of her own creation, SARS-CoV-2, changing a few nucleotides now and again to mimic the “random mutations” that regularly occur in nature.

She could easily have completed the “data entry” part of her task in a day, since all she was doing was entering in a string of letters alternating between the four nucleotides, A, U, G, and C. And coronaviruses contain less than 30,000 different nucleotides.

This article leans heavily on the premise that 'sars-cov-2' is an engineered virus, apparently they think it was accidentally released, apparently further near the end of the article that Dr. Fauci didn't apparently know (he would be in on it along with other WHO and GPMB board members also, whether 'sars-cov-2' is real or fake) they had created this 'virus' otherwise he should have known better than to support funding for the bat coronavirus surveillance and research program et al. Personally I don't buy that it has actually been “released” un-intentionally or intentionally or that it may actually exist outside of a theoretical construct. However this article to me seems to support my notion that 'sars-cov-2' genome has been created by a theoretical construct, and not being related by much of a phylogenetic traceable lineage to any other known coronaviruses (I don't think most people realize how many different coronaviruses there are, one paper said at least 500 different ones known I think).

Here they point out that the genome for RaTG13 was not published until January 27th 2020 and therfore only after the 'sars-cov-2' genome was published on January 12th,
Dr. Shi registered her new virus on January 27th, 2020, with the National Center for Biotechnology Information (NCBI) of the U.S. National Institutes of Health, the customary repository for such information. She called it RaTG-13, Ra for Rhinolophus affinis, the Latin name of the Intermediate Horseshoe Bat, and 13 for 2013, the year she supposedly “discovered” it.

A lot of people have been taken in by Dr. Shi’s clever “discovery,” which is looking more and more like a forgery. No one else has independently verified its existence. No other lab has a sample of it, and no one else has ever sequenced it. And of course, they likely never will, because more and more evidence suggests that it exists only in a string of letters on her computer.

In the “Reply to Science Magazine” interview the interviewer asking Dr. Shi about a virus (BtCoV/Ra4991, described in 2016 by only one protein but was 100% homologous to that protein in RaTG13), Dr. Shi stated that BtCoV/Ra4991 was the bat sample number and the virus was named RaTG13 (Ra is abbreviation and refers to the bat family and species), and in the same interview admitted that that was the only sample RaTG13 came from and was only 'sequenced” and not “isolated”.

So this article states there are other papers from the Chinese virology community that came out as 'evidence' that RaTG13 helps prove that 'sars-cov-2' couldn't have been “engineered” or a “recombination” of multiple viruses supposedly 'proving' a natural origin,
It was a brilliant scheme, and it almost succeeded. The Chinese virology community—following new, strict Party guidelines—has published a flurry of studies suggesting that the existence of RaTG-13 proves that SARS-CoV-2 came from nature. They claim that other “first cousins” of the China Coronavirus will soon be found to exist in nature if we just keep looking. They do genomic analyses showing that RaTG-13 and SARS-CoV-2 are 96% identical throughout the whole sequence of the viral genome. They calculate that the two share a common ancestor a few decades back.

Actually, the only thing the two virus genomes have in common is Dr. Shi herself, who engineered the one and appears to have fabricated the other.

As clever as Batwoman is, however, she did not commit the perfect deception. She left behind a few key clues that reveal, as clearly as fingerprints on a murder weapon, what she was up to.
So the article goes on to show with another contributor's analysis that genome of 'sars-cov-2' as presented shows too many un-probable and likely un-natural mutation events for the time frame suggested for it's origin/evolution from a common SARS-CoV ancestor virus:

One blogger, writing at Nerd Has Power, has brilliantly teased these findings out of the data. The blogger’s efforts can be read here if you have a free afternoon and a strong math background. I offer here a summary of one of the blogger’s critiques, in the hope of making the blogger’s general line of argument accessible to the layman. Because he published his raw data, I and others have been able to check and verify his work. I offer here a summary of one of the blogger’s critiques, in the hope of making the blogger’s general line of argument accessible to the layman.

An Impossible Ratio: As viruses evolve, they mutate. That is to say, one of the four nucleotides is randomly replaced by another. Most of these random mutations do not produce changes in the amino acids that make up the protein. Such mutations are called “synonymous”, since the three-nucleotide “codon” still codes for the same amino acid despite the change. Like a synonym in a thesaurus, it “looks” different but “means”—in terms of the amino acid and resulting protein—the same thing.

But then there are “non-synonymous” mutations. These are mutations that do change the resulting amino acid and hence the configuration of the resulting protein. In nature, the ratio of synonymous to non-synonymous is approximately 5:1.

Here’s where Dr. Shi got into trouble. When typing in the genomic sequence of her “discovery” she made way too many non-synonymous changes at the beginning. Then, one-third of the way through the sequence, she apparently realized her error. After that, she made way too few non-synonymous changes. So while the entire genome has the expected 5:1 ratio, there are stretches where the ratio is closer to 2:1, and other long stretches where it is as high as 44:1.

Nature’s mutations are random. Dr. Shi’s “mutations” are not. Dr. Lawrence Sellin has calculated that the odds that her “mutations” occurred naturally in just one area—the critical spike protein–at almost ten million to one.

If RaTG-13 is merely a strategic deception, as I believe it is, then what real coronavirus did Dr. Shi enhance in “Gain-of-Function” research to create the highly infectious SARS-CoV-2? Evidence suggests that a coronavirus from a People’s Liberation Army biolab may have played this role.

They take this analysis of the 'sars-cov-2' genome for the ratio of synonymous/non-synonymous mutation ratio (compared to SARS-CoV related CoV's I think) as evidence that the genome (and therefore the virus) RaTG13 is a fabrication, but why not take that further to suggest that 'sars-cov-2' is also a fabrication? A theoretical genome and no real virus? Perhaps they believe that Shi's paper regarding the alleged isolation of '2019-nCoV' is legit and that it is an engineered virus, really loose and killing people all over the world in 2020; but they would have to ignore all the other lies and fabrications and fakery of this entire psyop too, the way I see it.

So the other very interesting critique of Shi's paper I will look at before cutting off this huge post for further look at this paper later is this one,

Major Concerns on the Identification of Bat Coronavirus Strain RaTG13 and Quality of Related Nature Paper
Authors: Xiaoxu Sean Lin, Global Health Knowledge Exchange Inc., Silver spring, MD, U.S.A.Shizhong Chen, Genestitute, San Diego, CA, U.S.A
(NOT PEER-REVIEWED | Posted: 5 June 2020)
https://www.preprints.org/manuscript/202006.0044/v1

Not going to post a whole lot of this critique, it is very similar to the critique I posted in the last post from Monali C. Rahalkar and Rahul A. Bahulikar. I downloaded the pdf file, and here is the most interesting portion for me (beginning on page 4 of their critique), it seems to agree with me about the validity of the reverse de novo assembly/sequencing of the 'sars-cov-2' genome from BALF samples as opposed to doing the de novo sequencing from already prepared and “isolated/purified” virus stock samples, and therefore calling into question also whether RaTG13 is actually a real virus (since not “isolated”/no saved frozen samples) and calls into question the validity of at least “isolation” of a new virus presented as 'sars-cov-2' from probable bat origin, they seem to suggest to me that if they really did “isolate” any particular coronavirus at all it may have been faultily identified/characterized/sequenced, and therefore the original samples of RaTG13 (if actually exists, which Dr. Shi seemed to say in her interview that the sample it was detected from had been completely used up and never "isolated" from) and I would say further they indicate that the so-called stocks of 'sars'-cov-2' also both need to be re-examined by independent trustworthy experts.

This reminds me very much that the CDC in Atlanta GA did not have “isolated/purified” stocks of 'sars-cov-2' with which to test their PCR method against and if it was allegedly “isolated” as Dr. Shi's paper claims that is not validly tenable scientifically in my view that no such stocks were available to the CDC in the U.S., either they could have obtained them from Chinese sources (if legit) or could have repeated the procedure the Chinese used and “isolated” their own stocks from 'alleged' known 'covid-19' patients in the U.S. in order to have such viral stocks available, as I continue to say this is a huge red flag. And I'm certainly not saying that either one of these critiques, that any of the authors thereof are agreeing with me that any of their critique is proof that 'covid-19' is a fake psyop but I feel I can rightfully claim that if at least some of what they are saying is true and accurate it still lends support for my opinions and stances about 'covid-19' whether they had any such intentions or not.
Concerns on methodology, data quality and experiment procedures

Shi’s Nature paper1 mentioned that “Of the 10,038,758 total reads—of which 1,582 total reads were retained after filtering of reads from the human genome—1,378 (87.1%) sequences matched the sequence of SARSr-CoV (Fig. 1a). By de novo assembly and targeted PCR, we obtained a 29,891-base-pairCoV genome that shared 79.6% sequence identity to SARS-CoV BJ01 (GenBank accession number AY278488.2). High genome coverage was obtained by remapping the total reads to this genome (Extended Data Fig. 1). This sequence has been submitted to GISAID (https://www.gisaid.org/) (accession number EPI_ISL_402124).”

However, the methods described here to obtain full SARS-CoV-2 sequence have major flaws. First, characterization of novel viruses from patient samples using next-generation sequencing (NGS) technology must overcome the challenges posed by the high degree of genetic diversity observed across most virus families, especially for RNA viruses. Due to the error rate in RNA viral replication, what existed in a patient sample are usually viral quasispecies or mixed populations.Therefore, the method of random sequencing plus de novo assembly used in this study should only be used as the initial characterization. What is needed is to redo the NGS using the isolated virus stock so that the volume of raw reads related to target sequence would be significantly enhanced15. Then, reference assembly (using a SARS-CoV strain or Bat SARS-like CoV strain as reference) can be applied to obtain comprehensive coverage of the full viral genome with ample depth. Therefore, in this study, using only 1,378 reads from random amplification to conduct de novo assembly and getting near complete genome coverage (as shown in Extended Data Fig.1) for such a large RNA virus genome (near 30K in total length) isbeyond miracle.

Meanwhile, for regions with high chance of mutations such as spike protein open reading frame, very deep coverage of raw reads is often needed to ensure the accuracy of sequencing data. Random sequencing from patient samples would not work for genome regions with high variations, yet the paper did not mention any extra efforts being applied to address such concerns. The accuracy of the full genome sequences obtained in this study should be seriously challenged.

In the “Extended Data Fig. 6: Isolation and antigenic characterization of 2019-nCoV” of this paper1, it did mention carrying out“metagenomics analysis of supernatants from Vero E6 cell cultures”. Then, if NGS was conducted using viral supernatants from cell culture, the authors need to explain why those sets of NGS data was not presented in the paper, but instead NGS data from random sequencing of patient samples was presented. Therefore, the SARS-CoV-2 genome sequence submitted to the GISAID (accession number EPI_ISL_402124) in this study needs to be verified for its quality.

Meanwhile, Shi’s Naturepaper1mentioned that “four more full-length genome sequences of 2019-nCoV (WIV02, WIV05, WIV06 and WIV07) (GISAID accession numbers EPI_ISL_402127–402130) that were more than 99.9% identical to each other were subsequently obtained from four additional patients using next-generation sequencing and PCR (Extended Data Table 2).”However, since these full-length genome sequences were obtained without isolating the viruses from the patient samples(as explained in the footnote for Table 2)1, the quality of the genome sequences could be compromised. To ensure the quality of data submitted to GISAID, Zhengli Shi team should provide raw NGS reads to open platforms so that other scientists could review and re-analyze the raw sequencing data related to these important COVID-19 patient samples.
Meanwhile, on the official website of Wuhan Institute of Virology, the Institute leadership published an open letter to all its staffs and graduate students on February 17, 2020. In this open letter, it stated that the SARS-CoV-2 strain was isolated on January 5, 2020 and its full genome sequence was obtained as early as January 2, 202016. However, regarding the procedures for viral isolation, the Nature paper1also described in the Method section that “the culture supernatant was examined for the presence of virus by qRT–PCR methods developed in this study”. This indicated that the viral isolation could NOT be initiated at the same time with genome sequencing experiments from patient samples, because the qRT-PCR experiments would need specific primers and probes that could only be designed and produced after the genome sequencing was completed. This would suggest that Shi’s team only had as little as 2-3 days to obtain the viral isolate. Therefore, the experiment procedures described in this paper were surely rushed and needs further validation.

Conclusion:


In addition, there have been no studies on RaTG13’s infectivity in bat/human cells or in animal models, its interactions with antibodies or antiviral drugs. There is lack of understandings on RaTG13’s virulence, transmissibility, pathogenicity, immune epitopes, immune evasion mechanism, etc. This was because WIV did not isolate the RaTG13 virus and does not have any related viral stocks, if the statement from Dr. Yanyi Wang (the director of WIV) in a recent TV interview17 was accurate.

Therefore, a careful examination of the related RaTG13 samples and raw data sets of its genome sequencing are warranted to exclude any possibilities of errors or the potential co-infection of two different strains of coronavirus.
And the authors need to clearly explain the relationship between RaTG13 and BtCoV/4991, whether they were the same strain or two closely related strains. This paper was rushed to make a premature connection between bat coronavirus and SARS-CoV-2, drawing a potential bat origin scenario to support SARS-CoV-2 zoonotic transmission from bat to human. However, this connection was based on a potential bat coronavirus strain RaTG13, that may not truly exist, considering its key information missing: such as no related bat sample description, no sequencing procedure details published, confusion/identity issue with BtCoV/4991strain, unusual sequence features, no viral isolation and related characterization, etal.

In light of these concerns, we call for the retraction of this Nature paper1 to further verify the sequencing data, patient sample collection date and provide more information regarding the origin, identification and characterization of this BatCoV RaTG13. Proper verification should involve Dr.Zhengli Shi sending the RaTG13and BtCoV/4991-related bat samples to other non-collaborating laboratories to be analyzed independently. And this Nature paper1 should be cautious on making the “probable bat origin” hypothesis before RaTG13 existence could be confirmed.
 
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