Thanks for this. I've been researching "early spread" for 2 1/2 years and you are the first person who has even mentioned many of the same presumed cases I have. For example, you highlight the cases of Tim and Brandie McCain of Sylaucauga, Alabama. I sent my exclusive feature story on the McCains to about 30 news organizations and none would run it ... until finally Tracy Beanz at uncoverDC.com ran it.
The info on the deers and the case where the birth certificate was later revised were not known to me. Good luck with your research. I don't think many of us think "early spread" is a big deal, but it's actually a huge deal.
FYI, Santa Clara’s Patricia Dowd died Feb 6, 2020 in what CDC at the time classified as the nation’s first community spread case, after she posthumously tested positive for COVID-19 upon autopsy samples being sent to CDC. She worked for a Silicon Valley chip company and had been in China in the fall of 2019. She travelled on an airplane from the US northwest in Jan 2020. Would assume her coworkers were going to China a lot. Apparently her sister worked at a local hospital and reportedly treated covid patients. That was in local press reports. Left out of CDC’s MMWR. Always wondered why.
I posted a link to this article on Twitter, and received a lengthy 15-tweet response from "Ema Nympton" (@EmaNymton90) which I'm sharing here, since this is where it belongs.
[BEGINS]
I fully acknowledge that there were likely infections in the USA in Nov 2019. I also have contacts that report bad sickness in that time, and a +antibody test in early 2020.
However, my issue is linking it to EVALI.
1st- Ground glass opacities were known well before Covid-19.
Lots of things cause them, a series of people with pneumonia and GGOs, is not diagnostic of COVID, but it is certainly a big coincidence worth looking at. The timing, summer of 2019, is suspicious.
Vaping-Related Acute Parenchymal Lung Injury: A Systematic Review - PubMed 2012, but the main body hit summer 2019 - a temporal correlation. However, there's also a temporal correlation with the WIV taking its database down in September 2019 - which is an even stronger correlation, that seems to have a mutually exclusive explanation.
I keep coming back to: why was it first noticed in Wuhan, and not some 3rd party.
Speculation that the ancestral A-B precursor was less pathogenic (as opposed ot less transmissible), is speculative. We see when many viruses first start infecting humans, they are poorly transmissible, but cause very severe infections. MERS, SARS-1, the Mojiang miner sickness, etc
There are a whole lot of coincidences pertaining to Wuhan, but few pertaining to EVALI.
Of course, we could link US researchers to the WIV, but this is already quite similar to my current view of the pandemic as most likely resulting from a lab leak from a joint US-WIV research project. All that changes is the site of spillover. However, it seems the WIV kept its isolates there, and expts were done there because it was easier.
I can't rule out synthesizing a virues from a sequence obtained from China (or Laos).
I also can't rule out a September 2019 spillover in China, and a spread in the USA (and Europe) because of all the contacts with China.
Wuhan, if it was the origin, would presumably have the highest infection rate, given it started there first- the most time for the virus to spread (plus a very high poputation density), and adapt into lineage B.
I note D614G seems to have originated outside China, but the early hardest hit area of Italy was a region with many chinse immigrants that work in sweatshop like conditions (ideal for virus spread), with links to Wuhan as well.
However, coming back to the USA:
The one blood study showed evidence of infection in the earliest samples. It is inexcusable to not look farther back than Dec 2019.
I don't know about the CDC's primers, but while the CDC/FDA can dictate what is used for a diagnostic test for a patient, they can't dictate what a research group uses to analyze samples for a science paper. Also the multiple targets means I don't put much weight in the idea of a virus that was invisible to the early PCR tests in the USA.
I think hiding this would require a massive conspiracy that the USA doesn't have the infrastructure to implement.
I also think there has been an abhorrent lack of effort/resources put into dating the start of the outbreak in the USA.
I can't really explain why there hasn't been a proper retrospective study in the USA. There should be. Maybe its covering for EVALI, maybe its covering for Wuhan and a Sep 2019 incident, maybe it is something else entirely - I don't know.
I whole-heartedly support an effort to investigate the 2019 EVALI cases in the USA, and the start of the outbreak in the USA (or the rest of the world in genereal).
I just want to note that earlier circulation elsewhere does not automatically clear the WIV.
IMO, these results are a bit hard to reconcile with the US blood study, and the first lab-confirmed french patient in December with no travel history, while the Italian studies with nested PCRs and simple IgG reactivity are much weaker. [ENDS]
In addition, “Early viruses are more severe” is only an experience-based proposition. This is largely dependent on the route of infection (e.g. gastrointestinal or cutaneous infections may have been totally invisible if there is no lower respiratory tract infection) and does not necessarily apply in all viruses. For example, the Vaccinia virus is not adapted to humans and it don’t cause much pathogenesis at all. It is used in smallpox vaccination. Also, WIV and EHA in the U.S. are joint programs. The WIV may remove their database if a virus from the joint program leaked, regardless of where as it will always implicate both.
Not necessarily. The LRT may not have been unlocked without the virus learns to evade the two MiRNAs that are highly abundant in the human lungs and LRT. Early SARS-CoV-2 incidents (WA1, Wuhan but especially pandemic B.1) are characterized by the simultaneous infection of both the upper and the lower airways— https://www.sciencedirect.com/science/article/pii/S0092867420306759 where even recombinantly generated SARS-CoV-2 (WA1) were found to infect mostly the upper airways, and then only sometimes the lower airways. Infectivity is the greatest in the HNE (nasal epithelium) and LAE (tracheal and bronchial epithelium), and with SAE (lung causing pneumonia) having the least infectivity. The SARS and MERS norm unfortunately don’t apply for SARS-CoV-2. When AT-1/AT-2 infection is blockaded by MiRNA interference to the ORF1ab it left only the upper respiratory tract for respiratory infections— https://pubmed.ncbi.nlm.nih.gov/33009246/ where an anagelsic (sickness-suppressing) effect from the coincidental presence of RRxR in the S1-S2 junction (it is found to replace a more efficient RRXRR or RRRXRR FCS when CoVs adapts to cell cultures https://journals.asm.org/doi/10.1128/JVI.00074-08 as it confer binding to cell surface heparan sulfate in cultured cells submerged in liquid media, allowing more efficient attachment and cellular entry) mean that most non-pulmonary infections were completely asymptomatic especially in the early pandemic, with many patients not showing even ILI if the patient does not then later develop signs of pneumonia in Chest CT imaging. (if performed, most of even such patients would nevertheless still have only mild symptoms, and would eventually survive)
It takes some time for the virus to begin evading MiRNAs that were expressed in the respiratory tract for efficient replication and respiratory-centered pathogenesis/transmission, allowing the build-up of a highly infectious wave and generate detectable severity and lethality. Then it have to learn that too much replication the LRT is bad for long-term transmission and survival.
So it begin with low infectivity in the URT with skin manifestations and no infectivity in the LRT (prepandemic, RRXR exist but before MiR-146b and MiR-200c evasion gained on nt position 9400-0425 and 12094-12119 https://www.sciencedirect.com/science/article/pii/S0048969721012651), then in Wuhan it gained high infectivity in both the URT and the LRT (with higher infectivity in the URT compared to the LRT, as indicated in the recombinant generation study) by gaining evasion to MiR-146b and MiR-200c (and possibly being assembled together with a N protein CDS that make it replicate better such as with changes to phosphorylation of the N), before in later variants S protein changes abrogate LRT replication while maintaining or even greater enhancing replication in the URT.
The other problem is that in the NYC paper, where the N protein is omitted from the test, there were in deed circulation before the first declared pandemic there—the first sample they take that is fresh (remember that influenza test residual samples are nucleotide samples that were saved after a PCR test—storing them at 4 degree C for more than 2 days (prior to transfer, before commands made demanding them) is enough to destroy the RNA and cause negativity in the PCR test) already yielded an positive PCR result for the E protein CDS. Genomes were then recovered, reads from which indicated N protein mutations on the qRT-PCR primers that would lead to an escape with the more stringent annealing conditions. This indicate that yes, private researches were looking. And also unfortunately, when they looked, after the time when samples were no longer available before, their first fresh sample indicate that SARS-CoV-2 is already there.
Specified that “ RPN specimens were stored at +4 °C for 7 days after clinical testing. These residual RPN specimens were shipped weekly to the centralized clinical microbiology laboratory and frozen at −80 °C until thawed for pooling. RPN pools were aliquoted and stored at −80 °C until testing. Genomes recovered from RPN pools in this study were compared to later sequences obtained from individual clinical specimens from cases that tested positive for SARS-CoV-2 in MSHS once testing became more widely available. Details on testing using the aforementioned systems were previously described4.”
Yes these were stored in clinics for up to a week prior to collection and testing.
“ An important feature of this study is that we were able to begin retrieval and banking of RPN specimens from our laboratories immediately following the 8 January 2020 CDC Health Alert Network advisory regarding the outbreak of pneumonia of unknown etiology, later identified as COVID-19. Due to space constraints, most clinical laboratories are unable to store negative specimens for more than 1 week.”
The samples were retrieved as soon as possible, and up to 1 week is allowed in storage there. This time the samples were at +4 degrees C. They were RNA samples that were left over after a negative pathogen PCR test.
The problem with the Italian strain is that it is incapable of causing ILI-like symptoms due to the lack of a Wuhan-like N protein CDS. This prevents it from showing on qRT-PCR (and the fact that the qRT-PCR tests themselves require a high level of viral RNA presence, something that is difficult to achieve with stored samples as RNA tends to degrade over time), and likely incapable of causing symptomatic respiratory tract infections. However, it may remain capable of causing measles-like skin rashes and may have been specifically enriched there. As RNA degradation is only applicable to the original RNA within the samples, the total negativity of the control specimens rules out contamination as a source of the positive RNA results when a more sensitive test is used. The commercial PCR used the RdRp, E and the N, and standards in the test indicate that there must be either RdRp or E and one N or both of the N positive to rule the sample positive. This ran right into the problem which is that the SARS-CoV-2 Wuhan-specific N protein by itself may have not been present in the prepandemic strains, rendering it invisible to such qRT-PCR tests. Another problem with influenza surveillance samples are the fact that Influenza samples tends to be stored in lysis buffer and what that is made available for testing is the residual RNA after an influenza qRT-PCR test—where measles tests may have been different where the swab is stored in transport media and where the thing to be tested is the leftover transport media after the swab itself or an aliquot of the urine is used in the test. As such, the measles sample may have been better preserved (with good BioBank support, proper refrigeration) compared to the hospital conditions where the influenza samples were stored—which were already lysed at collection as influenza is highly transmissible even in immunized population, compared to measles which is stopped by early childhood vaccination. They also have different BSL levels as influenza strains-of-concern is BSL-3, meaning that intact virus may not be stored in certain BioBanks, and lysed samples only. This mean that the influenza surveillance itself likely have already degraded for sample positivity. This is evident since they even claim that the February 2020 samples were negative—samples from February 2020 which partially overlap with the official timeline of SARS-CoV-2 first case detection in Lombardy (sample collection did not stop until 9 days after first declared case), and a month after the official first detection in Rome. This would indicate that the samples were probably not properly stored for this influenza surveillance test, as much of the samples were taken after the official pandemic have already arrived in Italy (and supposedly included pneumonia case samples, which as in the postpandemic sampling period (02/2020) should have captured a true case with the supposed specific manifestation in pneumonia).
As for Switzerland and Toronto, they seems to be a little far away from France, Italy or Ontario……
In fact, from contacts traced to official cases taken from Lombardy (after they begin PCR testing from the first China-linked case of “ARDS with unknown etiology” in 20/02/2020), PCR tested beginning in 21/02/2020, cases and case contacts that had symptoms onset as early as 30/01/2020 have been found, with more than 440 PCR positive cases contact traced from reported cases before 20/01/2020. (The only way a case could be suspected and tested without a link to China or a known suspected or confirmed case at that point of time is for the case to have ARDS, and is a HCW that treated patients with SARI of unknown etiology—curiously, said patients are not classified as suspected cases even if the HCW is.) (unfortunately contact tracing does not always guarantee direct transmission as the cause. Most of the time they ended up picking up community cases that were caught into the highly expansive contact tree by accident, and where no phylogenetic link between the virus genome recovered and that is found in China at that point, should a sequencing of the virus sample is performed, is found. This is especially prevalent in Germany and France in addition to Italy itself.)
Most of these contact traced cases were collected with no respect to symptoms and were tested between 21/02/2020 and 25/02/2020. As such, ILI surveillance should have readily caught these cases (contact traced cases and any potential community transmission generated from the contact trees of officially reported cases, which sets the latest limit where ILI surveillance sampling should begun detecting SARS-CoV-2 from their collected respiratory samples under the null hypothesis) and return positive samples from sampling in 02/2020. However, the ILI surveillance paper https://ncbi.nlm.nih.gov/pmc/articles/PMC7988398/ failed to detect positives even from this time period (in fact, none of the samples they tested were “positive”), which indicate that unfavored storage condition (such as the use of lysis buffer during sampling, or the storage of already-extracted leftover RNA in stead of biological samples in their original state) likely degraded all the RNA within these ILI surveillance samples—such that not even the expected positivity under the null hypothesis (As least the February 2020 ILI samples should turn positive even under the null hypothesis of taking only the official timeline of Covid-19 case reports in Italy and contact tracing in Lombardy into account) show in the results. This is therefore unlikely to be indicative to a genuine absence of circulation as the results claimed, but only that the samples from ILI surveillance have degraded and amplification even from the post-pandemic fractions of their series were no longer possible.
This is a hypothesis, but there is a problem here https://www.biorxiv.org/content/10.1101/2022.10.13.512134v1.full which is that Omicron, with 3AA deletion between N1-R and N2-F, is attenuated independent of the Spike. The N protein is important for replication and pathogenesis and a bad N protein stop respiratory pathogenesis.
Here is where our understanding meets a lacunae. The Omicron experiment clearly tells us that attenuation happens with its backbone, where a deletion of the N happens. We also know that R203K:G204R lead to greater replication of the virus. The Nucleocapsid protein is an important structural component of Coronaviruses and the sequence of it directly affects the ability for the virus to replicate, as the N determines the level of shielding CoV genomic RNA receive from innate immune recognition and that infectious clones of CoVs are rescued with greater efficiency when the N protein is provided.
Mutations in the N protein is found to correlate with pathogenesis, so it is not unreasonable that some N protein variants may be attenuated in pathogenesis or lack respiratory tropism. However, the RdRp may have a greater role in controlling the respiratory tropism of SARS-CoV-2 in humans as it carried two human lung-specific MiRNA targets where the bat relatives were more restricted compared to SARs-CoV-2 itself due to additional mismatches on these 2 targets to the respective MiRNAs compared to that is found in bats.
Other changes to the N protein, such as ablation of its phosphorylation by GSK-3, also lead to attenuation of replication in the lungs. While this is performed by inhibiting of knocking out host GSK-3 in the experiment, Mutations on the GSK-3 target sites will cause a similar result, which can control the replication and respiratory virulence of the resulting viral genome.
" the fact that the qRT-PCR tests themselves require a high level of viral RNA presence, something that is difficult to achieve with stored samples as RNA tends to degrade over time"
If the original sample was positive from a few DPOS is very very easy to detect the RNA by qt-PCR, without using nested PCR it any of that stuff, and is done all the time
The problem here is that in the influenza tests not even samples that were taken a month after the official first case in Italy (and where 450+ retrospectively contact traced cases from officially recognized first Lombardy case, dating back to 30/01/2020 were found in the time period where the last influenza samples were collected (and where the PCR testing result is positive when they were screened in 20-25/02/2020)) contained positive qRT-PCR test results. This is highly unusual (as mild Covid-19 symptoms overlapped closely with ILI, and the sample series is supposed to contain all SARI samples in ICU from the region as well) as they indicated no sample tested positive—including samples taken in February 2020 (30% of all their samples, 478 ,came from ILI and SARI patients taken after the date of symptoms onset of the first officially contact traced positive patient from imported cases in Lombardy). This is extremely unlikely and most likely indicate that the samples (residual RNA lysates/extracts left an Influenza-specific qRT-PCR test) have already completely degraded that none of them contained amplifiable proportions of RNA inside. This is most likely because unlike measles where the swabs were stored in viral storage/transport media, and urine is a liquid by itself, and the original samples being tested, influenza samples are biologically hazardous so they have to be first inactivated through lysis (swabs immersed in lysis buffer and the non-hazardous pre-extracted RNA is stored) before they can be transported to labs and tested. This mean that influenza samples are much less stable than measles samples, resulting in total negativity even after the official start of the pandemic (and extensive evidence of community spread in even the restricted sample of cases contact traced to the “officially recognized” (where the “must be China linked” condition likely resulted in massive underdiagnosis) cases) in the sampled region.
As the influenza surveillance program runs independently from the Covid-19 screening program, and the paper itself did not specify any exclusion criterion on the direct or indirect contacts of officially recognized Covid-19 cases (by link to China, and by attending to SARI patients in a hospital) within Italy or Lombardy (where positive samples with symptoms onset up to 30/01/2020 were seen), February 2020 function as a positive control for the Influenza paper the same way “pandemic period (2021 samples)” function as a positive control for the measles paper.
No experiments verifying the sensitivity of the qRT-PCR tests were employed in the influenza paper, and as even under the null hypothesis of “only the official cases and those that were found by contact tracing from these cases and tested positive on the spot were real infections”, positivity is expected from samples taken in 02/2020 (a month after symptoms onset of the earliest of such a case, https://www.sciencedirect.com/science/article/pii/S1755436521000724 and up to 8 days after the first officially recognized case under the strict European CDC standards), a total negativity within this group is not expected even if the null hypothesis proves true.
Therefore the most likely cause of the specified results (0 positives from all 478 collected ILI and SARI samples, containing most if not all SARI samples taken from Lombardy in February 2020) in the influenza paper is that the tests they used were insufficiently sensitive to detect cases even after the beginning of the official pandemic within Italy and Lombardy—most likely cause is sample degredation or PCR inhibition from a component of the reagents used during the collection, processing, storage or amplification of the RNA samples in the Influenza surveillance program.
Some protocols involving the Influenza surveillance samples (e.g. using the swab itself after an initial RNA extraction for influenza test, as opposed to the UTM (Universal Transport Medium) and Urine used in the measles surveillance which were not subjected to lysis prior to RNA extraction for SARS-CoV-2 PCR testing), could also lead to the destruction of SARS-CoV-2 RNA before a test is conducted. Unlike influenza virus which replicated within the nucleus, and have an tightly bound NP protein around the viral RNA all the time during replication (which does not have to be displaced or spontaneously disassociate from the RNA during translation of its genes, as in CoV gRNA), attached to the membrane via the M2 protein; as well as HA proteins that binds to sugar residues (cotton swabs are composed of polysaccharides and contained several N-linked glycans);
Coronavirus gRNA replicates within the cytoplasm, and is only loosely bound to either the N or the M proteins as it needs to disassociate from the N and M in order to be translated by the cellular ribosome. This mean that Unlike influenza virus RNA which can sometimes survive multiple extractions, Coronavirus RNA is completely eluted within 1 extraction in the majority of protocols involved in viral RNA extraction, leaving nothing to be tested when these influenza surveillance samples were extracted a second time for SARS-CoV-2 testing—to the point that even samples taken after the first officially recognized cases were contact traced and tested positive, ~30% of all their samples involving all SARI samples taken from that era, tested negative. If it is already after the pandemic yet your samples were still negative, most likely your protocol prevented a positive test.
Thanks for this. I've been researching "early spread" for 2 1/2 years and you are the first person who has even mentioned many of the same presumed cases I have. For example, you highlight the cases of Tim and Brandie McCain of Sylaucauga, Alabama. I sent my exclusive feature story on the McCains to about 30 news organizations and none would run it ... until finally Tracy Beanz at uncoverDC.com ran it.
The info on the deers and the case where the birth certificate was later revised were not known to me. Good luck with your research. I don't think many of us think "early spread" is a big deal, but it's actually a huge deal.
you missed the best part of the latest deer paper:
https://pubmed.ncbi.nlm.nih.gov/36357713/
no immune response!
https://youtu.be/tTsOn-4DLmM?t=2238
Your first date, I think you mean December 2019.
FYI, Santa Clara’s Patricia Dowd died Feb 6, 2020 in what CDC at the time classified as the nation’s first community spread case, after she posthumously tested positive for COVID-19 upon autopsy samples being sent to CDC. She worked for a Silicon Valley chip company and had been in China in the fall of 2019. She travelled on an airplane from the US northwest in Jan 2020. Would assume her coworkers were going to China a lot. Apparently her sister worked at a local hospital and reportedly treated covid patients. That was in local press reports. Left out of CDC’s MMWR. Always wondered why.
Excellent research. Any hope of re-instatement on Twitter. We miss you
Very good.
I posted a link to this article on Twitter, and received a lengthy 15-tweet response from "Ema Nympton" (@EmaNymton90) which I'm sharing here, since this is where it belongs.
[BEGINS]
I fully acknowledge that there were likely infections in the USA in Nov 2019. I also have contacts that report bad sickness in that time, and a +antibody test in early 2020.
However, my issue is linking it to EVALI.
1st- Ground glass opacities were known well before Covid-19.
Lots of things cause them, a series of people with pneumonia and GGOs, is not diagnostic of COVID, but it is certainly a big coincidence worth looking at. The timing, summer of 2019, is suspicious.
According to this, there were earlier cases https://pubmed.ncbi.nlm.nih.gov/32442559/ as early as
pubmed.ncbi.nlm.nih.gov
Vaping-Related Acute Parenchymal Lung Injury: A Systematic Review - PubMed 2012, but the main body hit summer 2019 - a temporal correlation. However, there's also a temporal correlation with the WIV taking its database down in September 2019 - which is an even stronger correlation, that seems to have a mutually exclusive explanation.
I keep coming back to: why was it first noticed in Wuhan, and not some 3rd party.
Speculation that the ancestral A-B precursor was less pathogenic (as opposed ot less transmissible), is speculative. We see when many viruses first start infecting humans, they are poorly transmissible, but cause very severe infections. MERS, SARS-1, the Mojiang miner sickness, etc
There are a whole lot of coincidences pertaining to Wuhan, but few pertaining to EVALI.
Of course, we could link US researchers to the WIV, but this is already quite similar to my current view of the pandemic as most likely resulting from a lab leak from a joint US-WIV research project. All that changes is the site of spillover. However, it seems the WIV kept its isolates there, and expts were done there because it was easier.
I can't rule out synthesizing a virues from a sequence obtained from China (or Laos).
I also can't rule out a September 2019 spillover in China, and a spread in the USA (and Europe) because of all the contacts with China.
Wuhan, if it was the origin, would presumably have the highest infection rate, given it started there first- the most time for the virus to spread (plus a very high poputation density), and adapt into lineage B.
I note D614G seems to have originated outside China, but the early hardest hit area of Italy was a region with many chinse immigrants that work in sweatshop like conditions (ideal for virus spread), with links to Wuhan as well.
However, coming back to the USA:
The one blood study showed evidence of infection in the earliest samples. It is inexcusable to not look farther back than Dec 2019.
I don't know about the CDC's primers, but while the CDC/FDA can dictate what is used for a diagnostic test for a patient, they can't dictate what a research group uses to analyze samples for a science paper. Also the multiple targets means I don't put much weight in the idea of a virus that was invisible to the early PCR tests in the USA.
I think hiding this would require a massive conspiracy that the USA doesn't have the infrastructure to implement.
I also think there has been an abhorrent lack of effort/resources put into dating the start of the outbreak in the USA.
I can't really explain why there hasn't been a proper retrospective study in the USA. There should be. Maybe its covering for EVALI, maybe its covering for Wuhan and a Sep 2019 incident, maybe it is something else entirely - I don't know.
I whole-heartedly support an effort to investigate the 2019 EVALI cases in the USA, and the start of the outbreak in the USA (or the rest of the world in genereal).
I just want to note that earlier circulation elsewhere does not automatically clear the WIV.
Lastly, there are these studies:
https://ncbi.nlm.nih.gov/pmc/articles/PMC7365111/
https://pubmed.ncbi.nlm.nih.gov/36340052/
https://ncbi.nlm.nih.gov/pmc/articles/PMC7988398/
IMO, these results are a bit hard to reconcile with the US blood study, and the first lab-confirmed french patient in December with no travel history, while the Italian studies with nested PCRs and simple IgG reactivity are much weaker. [ENDS]
In addition, “Early viruses are more severe” is only an experience-based proposition. This is largely dependent on the route of infection (e.g. gastrointestinal or cutaneous infections may have been totally invisible if there is no lower respiratory tract infection) and does not necessarily apply in all viruses. For example, the Vaccinia virus is not adapted to humans and it don’t cause much pathogenesis at all. It is used in smallpox vaccination. Also, WIV and EHA in the U.S. are joint programs. The WIV may remove their database if a virus from the joint program leaked, regardless of where as it will always implicate both.
Re: "does not necessarily apply in all viruses"
I agree, but my point is that it is speculation that an earlier variant would have been milder.
It took quite a while before we saw replication shift away from the LRT to the URT and nasal cavities.
SC1 replicated in the LRT
Occam's razor suggests the earliest variants replicated in the LRT
Not necessarily. The LRT may not have been unlocked without the virus learns to evade the two MiRNAs that are highly abundant in the human lungs and LRT. Early SARS-CoV-2 incidents (WA1, Wuhan but especially pandemic B.1) are characterized by the simultaneous infection of both the upper and the lower airways— https://www.sciencedirect.com/science/article/pii/S0092867420306759 where even recombinantly generated SARS-CoV-2 (WA1) were found to infect mostly the upper airways, and then only sometimes the lower airways. Infectivity is the greatest in the HNE (nasal epithelium) and LAE (tracheal and bronchial epithelium), and with SAE (lung causing pneumonia) having the least infectivity. The SARS and MERS norm unfortunately don’t apply for SARS-CoV-2. When AT-1/AT-2 infection is blockaded by MiRNA interference to the ORF1ab it left only the upper respiratory tract for respiratory infections— https://pubmed.ncbi.nlm.nih.gov/33009246/ where an anagelsic (sickness-suppressing) effect from the coincidental presence of RRxR in the S1-S2 junction (it is found to replace a more efficient RRXRR or RRRXRR FCS when CoVs adapts to cell cultures https://journals.asm.org/doi/10.1128/JVI.00074-08 as it confer binding to cell surface heparan sulfate in cultured cells submerged in liquid media, allowing more efficient attachment and cellular entry) mean that most non-pulmonary infections were completely asymptomatic especially in the early pandemic, with many patients not showing even ILI if the patient does not then later develop signs of pneumonia in Chest CT imaging. (if performed, most of even such patients would nevertheless still have only mild symptoms, and would eventually survive)
It takes some time for the virus to begin evading MiRNAs that were expressed in the respiratory tract for efficient replication and respiratory-centered pathogenesis/transmission, allowing the build-up of a highly infectious wave and generate detectable severity and lethality. Then it have to learn that too much replication the LRT is bad for long-term transmission and survival.
So it begin with low infectivity in the URT with skin manifestations and no infectivity in the LRT (prepandemic, RRXR exist but before MiR-146b and MiR-200c evasion gained on nt position 9400-0425 and 12094-12119 https://www.sciencedirect.com/science/article/pii/S0048969721012651), then in Wuhan it gained high infectivity in both the URT and the LRT (with higher infectivity in the URT compared to the LRT, as indicated in the recombinant generation study) by gaining evasion to MiR-146b and MiR-200c (and possibly being assembled together with a N protein CDS that make it replicate better such as with changes to phosphorylation of the N), before in later variants S protein changes abrogate LRT replication while maintaining or even greater enhancing replication in the URT.
The other problem is that in the NYC paper, where the N protein is omitted from the test, there were in deed circulation before the first declared pandemic there—the first sample they take that is fresh (remember that influenza test residual samples are nucleotide samples that were saved after a PCR test—storing them at 4 degree C for more than 2 days (prior to transfer, before commands made demanding them) is enough to destroy the RNA and cause negativity in the PCR test) already yielded an positive PCR result for the E protein CDS. Genomes were then recovered, reads from which indicated N protein mutations on the qRT-PCR primers that would lead to an escape with the more stringent annealing conditions. This indicate that yes, private researches were looking. And also unfortunately, when they looked, after the time when samples were no longer available before, their first fresh sample indicate that SARS-CoV-2 is already there.
Normally, NPS swabs are frozen, not stored at 4C, it's what they do where I am
Citation?
For these, the specific article that worked with these NPS samples
https://www.nature.com/articles/s41467-021-23688-7
Specified that “ RPN specimens were stored at +4 °C for 7 days after clinical testing. These residual RPN specimens were shipped weekly to the centralized clinical microbiology laboratory and frozen at −80 °C until thawed for pooling. RPN pools were aliquoted and stored at −80 °C until testing. Genomes recovered from RPN pools in this study were compared to later sequences obtained from individual clinical specimens from cases that tested positive for SARS-CoV-2 in MSHS once testing became more widely available. Details on testing using the aforementioned systems were previously described4.”
Yes these were stored in clinics for up to a week prior to collection and testing.
“ An important feature of this study is that we were able to begin retrieval and banking of RPN specimens from our laboratories immediately following the 8 January 2020 CDC Health Alert Network advisory regarding the outbreak of pneumonia of unknown etiology, later identified as COVID-19. Due to space constraints, most clinical laboratories are unable to store negative specimens for more than 1 week.”
The samples were retrieved as soon as possible, and up to 1 week is allowed in storage there. This time the samples were at +4 degrees C. They were RNA samples that were left over after a negative pathogen PCR test.
The problem with the Italian strain is that it is incapable of causing ILI-like symptoms due to the lack of a Wuhan-like N protein CDS. This prevents it from showing on qRT-PCR (and the fact that the qRT-PCR tests themselves require a high level of viral RNA presence, something that is difficult to achieve with stored samples as RNA tends to degrade over time), and likely incapable of causing symptomatic respiratory tract infections. However, it may remain capable of causing measles-like skin rashes and may have been specifically enriched there. As RNA degradation is only applicable to the original RNA within the samples, the total negativity of the control specimens rules out contamination as a source of the positive RNA results when a more sensitive test is used. The commercial PCR used the RdRp, E and the N, and standards in the test indicate that there must be either RdRp or E and one N or both of the N positive to rule the sample positive. This ran right into the problem which is that the SARS-CoV-2 Wuhan-specific N protein by itself may have not been present in the prepandemic strains, rendering it invisible to such qRT-PCR tests. Another problem with influenza surveillance samples are the fact that Influenza samples tends to be stored in lysis buffer and what that is made available for testing is the residual RNA after an influenza qRT-PCR test—where measles tests may have been different where the swab is stored in transport media and where the thing to be tested is the leftover transport media after the swab itself or an aliquot of the urine is used in the test. As such, the measles sample may have been better preserved (with good BioBank support, proper refrigeration) compared to the hospital conditions where the influenza samples were stored—which were already lysed at collection as influenza is highly transmissible even in immunized population, compared to measles which is stopped by early childhood vaccination. They also have different BSL levels as influenza strains-of-concern is BSL-3, meaning that intact virus may not be stored in certain BioBanks, and lysed samples only. This mean that the influenza surveillance itself likely have already degraded for sample positivity. This is evident since they even claim that the February 2020 samples were negative—samples from February 2020 which partially overlap with the official timeline of SARS-CoV-2 first case detection in Lombardy (sample collection did not stop until 9 days after first declared case), and a month after the official first detection in Rome. This would indicate that the samples were probably not properly stored for this influenza surveillance test, as much of the samples were taken after the official pandemic have already arrived in Italy (and supposedly included pneumonia case samples, which as in the postpandemic sampling period (02/2020) should have captured a true case with the supposed specific manifestation in pneumonia).
As for Switzerland and Toronto, they seems to be a little far away from France, Italy or Ontario……
https://www.sciencedirect.com/science/article/pii/S1755436521000724
In fact, from contacts traced to official cases taken from Lombardy (after they begin PCR testing from the first China-linked case of “ARDS with unknown etiology” in 20/02/2020), PCR tested beginning in 21/02/2020, cases and case contacts that had symptoms onset as early as 30/01/2020 have been found, with more than 440 PCR positive cases contact traced from reported cases before 20/01/2020. (The only way a case could be suspected and tested without a link to China or a known suspected or confirmed case at that point of time is for the case to have ARDS, and is a HCW that treated patients with SARI of unknown etiology—curiously, said patients are not classified as suspected cases even if the HCW is.) (unfortunately contact tracing does not always guarantee direct transmission as the cause. Most of the time they ended up picking up community cases that were caught into the highly expansive contact tree by accident, and where no phylogenetic link between the virus genome recovered and that is found in China at that point, should a sequencing of the virus sample is performed, is found. This is especially prevalent in Germany and France in addition to Italy itself.)
Most of these contact traced cases were collected with no respect to symptoms and were tested between 21/02/2020 and 25/02/2020. As such, ILI surveillance should have readily caught these cases (contact traced cases and any potential community transmission generated from the contact trees of officially reported cases, which sets the latest limit where ILI surveillance sampling should begun detecting SARS-CoV-2 from their collected respiratory samples under the null hypothesis) and return positive samples from sampling in 02/2020. However, the ILI surveillance paper https://ncbi.nlm.nih.gov/pmc/articles/PMC7988398/ failed to detect positives even from this time period (in fact, none of the samples they tested were “positive”), which indicate that unfavored storage condition (such as the use of lysis buffer during sampling, or the storage of already-extracted leftover RNA in stead of biological samples in their original state) likely degraded all the RNA within these ILI surveillance samples—such that not even the expected positivity under the null hypothesis (As least the February 2020 ILI samples should turn positive even under the null hypothesis of taking only the official timeline of Covid-19 case reports in Italy and contact tracing in Lombardy into account) show in the results. This is therefore unlikely to be indicative to a genuine absence of circulation as the results claimed, but only that the samples from ILI surveillance have degraded and amplification even from the post-pandemic fractions of their series were no longer possible.
Citation for the claim that "the Italian strain" couldn't cause ILI symptoms because of its N sequence?
This is a hypothesis, but there is a problem here https://www.biorxiv.org/content/10.1101/2022.10.13.512134v1.full which is that Omicron, with 3AA deletion between N1-R and N2-F, is attenuated independent of the Spike. The N protein is important for replication and pathogenesis and a bad N protein stop respiratory pathogenesis.
I have seen other papers suggesting the pathogenicity difference lies in the E gene, or Nsp genes.
My problem here is that you are stating speculation as if it were fact
Here is where our understanding meets a lacunae. The Omicron experiment clearly tells us that attenuation happens with its backbone, where a deletion of the N happens. We also know that R203K:G204R lead to greater replication of the virus. The Nucleocapsid protein is an important structural component of Coronaviruses and the sequence of it directly affects the ability for the virus to replicate, as the N determines the level of shielding CoV genomic RNA receive from innate immune recognition and that infectious clones of CoVs are rescued with greater efficiency when the N protein is provided.
Mutations in the N protein is found to correlate with pathogenesis, so it is not unreasonable that some N protein variants may be attenuated in pathogenesis or lack respiratory tropism. However, the RdRp may have a greater role in controlling the respiratory tropism of SARS-CoV-2 in humans as it carried two human lung-specific MiRNA targets where the bat relatives were more restricted compared to SARs-CoV-2 itself due to additional mismatches on these 2 targets to the respective MiRNAs compared to that is found in bats.
https://www.medrxiv.org/content/10.1101/2021.02.17.21251933v2.full
Other changes to the N protein, such as ablation of its phosphorylation by GSK-3, also lead to attenuation of replication in the lungs. While this is performed by inhibiting of knocking out host GSK-3 in the experiment, Mutations on the GSK-3 target sites will cause a similar result, which can control the replication and respiratory virulence of the resulting viral genome.
" the fact that the qRT-PCR tests themselves require a high level of viral RNA presence, something that is difficult to achieve with stored samples as RNA tends to degrade over time"
If the original sample was positive from a few DPOS is very very easy to detect the RNA by qt-PCR, without using nested PCR it any of that stuff, and is done all the time
The problem here is that in the influenza tests not even samples that were taken a month after the official first case in Italy (and where 450+ retrospectively contact traced cases from officially recognized first Lombardy case, dating back to 30/01/2020 were found in the time period where the last influenza samples were collected (and where the PCR testing result is positive when they were screened in 20-25/02/2020)) contained positive qRT-PCR test results. This is highly unusual (as mild Covid-19 symptoms overlapped closely with ILI, and the sample series is supposed to contain all SARI samples in ICU from the region as well) as they indicated no sample tested positive—including samples taken in February 2020 (30% of all their samples, 478 ,came from ILI and SARI patients taken after the date of symptoms onset of the first officially contact traced positive patient from imported cases in Lombardy). This is extremely unlikely and most likely indicate that the samples (residual RNA lysates/extracts left an Influenza-specific qRT-PCR test) have already completely degraded that none of them contained amplifiable proportions of RNA inside. This is most likely because unlike measles where the swabs were stored in viral storage/transport media, and urine is a liquid by itself, and the original samples being tested, influenza samples are biologically hazardous so they have to be first inactivated through lysis (swabs immersed in lysis buffer and the non-hazardous pre-extracted RNA is stored) before they can be transported to labs and tested. This mean that influenza samples are much less stable than measles samples, resulting in total negativity even after the official start of the pandemic (and extensive evidence of community spread in even the restricted sample of cases contact traced to the “officially recognized” (where the “must be China linked” condition likely resulted in massive underdiagnosis) cases) in the sampled region.
As the influenza surveillance program runs independently from the Covid-19 screening program, and the paper itself did not specify any exclusion criterion on the direct or indirect contacts of officially recognized Covid-19 cases (by link to China, and by attending to SARI patients in a hospital) within Italy or Lombardy (where positive samples with symptoms onset up to 30/01/2020 were seen), February 2020 function as a positive control for the Influenza paper the same way “pandemic period (2021 samples)” function as a positive control for the measles paper.
No experiments verifying the sensitivity of the qRT-PCR tests were employed in the influenza paper, and as even under the null hypothesis of “only the official cases and those that were found by contact tracing from these cases and tested positive on the spot were real infections”, positivity is expected from samples taken in 02/2020 (a month after symptoms onset of the earliest of such a case, https://www.sciencedirect.com/science/article/pii/S1755436521000724 and up to 8 days after the first officially recognized case under the strict European CDC standards), a total negativity within this group is not expected even if the null hypothesis proves true.
Therefore the most likely cause of the specified results (0 positives from all 478 collected ILI and SARI samples, containing most if not all SARI samples taken from Lombardy in February 2020) in the influenza paper is that the tests they used were insufficiently sensitive to detect cases even after the beginning of the official pandemic within Italy and Lombardy—most likely cause is sample degredation or PCR inhibition from a component of the reagents used during the collection, processing, storage or amplification of the RNA samples in the Influenza surveillance program.
Some protocols involving the Influenza surveillance samples (e.g. using the swab itself after an initial RNA extraction for influenza test, as opposed to the UTM (Universal Transport Medium) and Urine used in the measles surveillance which were not subjected to lysis prior to RNA extraction for SARS-CoV-2 PCR testing), could also lead to the destruction of SARS-CoV-2 RNA before a test is conducted. Unlike influenza virus which replicated within the nucleus, and have an tightly bound NP protein around the viral RNA all the time during replication (which does not have to be displaced or spontaneously disassociate from the RNA during translation of its genes, as in CoV gRNA), attached to the membrane via the M2 protein; as well as HA proteins that binds to sugar residues (cotton swabs are composed of polysaccharides and contained several N-linked glycans);
Coronavirus gRNA replicates within the cytoplasm, and is only loosely bound to either the N or the M proteins as it needs to disassociate from the N and M in order to be translated by the cellular ribosome. This mean that Unlike influenza virus RNA which can sometimes survive multiple extractions, Coronavirus RNA is completely eluted within 1 extraction in the majority of protocols involved in viral RNA extraction, leaving nothing to be tested when these influenza surveillance samples were extracted a second time for SARS-CoV-2 testing—to the point that even samples taken after the first officially recognized cases were contact traced and tested positive, ~30% of all their samples involving all SARI samples taken from that era, tested negative. If it is already after the pandemic yet your samples were still negative, most likely your protocol prevented a positive test.