COVID-19 News: Yale Study Shockingly Finds That PBMC Myeloid Cells Harbors SARS-CoV-2 Genomes With Implications For Viral Persistence And Misc Dangers!

COVID-19 News: Scientists from Yale Medical School have made yet another alarming discovery that the human host’s blood mononuclear cells (PBMC) are able to harbor the SARS-CoV-2 genome with implications for viral persistence and dangers to various human organs over time.


 
Various different viruses infect circulating mononuclear cells to disseminate infection to diverse organs. Blood mononuclear cells (PBMC) are being intensively studied as immunologic and pathologic responders to the new pandemic SARS-CoV-2 virus (CoV19) but direct evidence showing CoV19 in these cells has not been published.
 
PBMC myeloid cells that take up residence in various organs can harbor viral genomes for many years in lymphatic tissues, skin and brain, and act as a source for re-infection and/or post-viral organ pathology.
 
In order to test if PBMC from acutely ill hospitalized patients contain viral nucleic acids, the study team from Yale first analyzed a standard short CoV19 nucleocapsid (NC) 72bp sequence. Because NC proteins protect the viral genome, the study team further analyzed longer (301nt) adjacent NC stretches by RNA/qPCR.
 
Shockingly in 2 of 11 patient’s PBMC, but not uninfected controls, longer NC sequences were positive as early as 2-6 days after hospital admission and were validated by sequencing.
 
 The presence of longer NC sequences indicates pathogenic fragments, or possibly the complete infectious virus, are carried by a rare population of monocytes, probably a subset of myeloid migratory cells. Predictably, such cells carried CoV19 to heart and brain with consequent late post-viral immune pathologies that are now evident.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2020.12.16.423113v1
 
It is known that certain acute lytic viral infections release free viral particles into the bloodstream where they are easily assayed with modern molecular techniques. However, a large variety of viruses travel within white blood cells such as retroviruses, e.g., HIV and flaviviruses, e.g., Dengue. https://pubmed.ncbi.nlm.nih.gov/31396206/
 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607932/
 
https://pubmed.ncbi.nlm.nih.gov/29106931/
 
Importantly certain viruses carried in white blood cells are associated with chronic disease. Poliovirus, a "neurotropic" enterovirus originally thought to spread directly through nerves, instead transits from gut to white blood cells to spleen, and only later infects brain. https://journals.sagepub.com/doi/abs/10.3181/00379727-79-19398
 
To date there are about 13 known different classes of DNA and RNA viruses that infect peripheral blood mononuclear cells (PBMC) that can be a reservoir for persistence, in addition to the infectious Creut zfeldt-Jakob Disease (CJD) agent. https://pubmed.ncbi.nlm.nih.gov/30231586/
 
https://pubmed.ncbi.nlm.nih.gov/349691/
 
Like HTLV1, a retrovirus that can be sequestered in brain myeloid microglia, the CJD agent in white blood cells progressively increases after primary infection of gut dendritic (myeloid lineage) cells to later show up in highly infectious myeloid microglia in brain. The CJD agent requires cell-to-cell contact for infection, probably via viral synapses, the same mechanism or conduit used for T to myeloid cell transmission of HIV. https://pubmed.ncbi.nlm.nih.gov/16239476/
 
Moreover, chronic HIV infection of brain microglia is linked to neurocognitive disorders and dementia, as is the very different ~20-25nm CJD infectious particle. These observations emphasize the general principle that infected migrating myeloid cells can take up residence in and perpetuate infection and chronic pathology in the brain and other organs. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1794316/
 
Importantly the presence of nucleocapsid CoV19 RNA, especially in migrating myeloid cells, might explain some of the ensuing brain and heart pathologies that were predictable and that have become increasingly evident during this COVID-19 pandemic.
 
The study team was aware that PBMC could contain extremely low CoV19 RNA due to the presence of the virus possibly limited to rare myeloid cell types.
 
Such cell types can be less abundant than the 1-2% dendritic cells in a PBMC population typically dominated by lymphocytes (70-90%). Many different myeloid cell-types are increasingly appreciated and classified, and many of them show wide phenotypic flexibility. https://pubmed.ncbi.nlm.nih.gov/28031342/
 
Furthermore, total cellular RNA is very low in PBMC, ~1/40th of that produced by epithelial and cultured tumor cells such as HeLa cells and NIH/3T3 cells.
 
According to the study team, there were 3 additional reasons to pursue this study:
 
-1)Coronaviruses are complex, and can elicit autoimmune responses that damage brain. For example, the mouse hepatitis coronavirus (MHV) induces a post-viral autoimmune demyelinating disease that is a model of Multiple Sclerosis, and other coronavirus strains, such as CoV19, might elicit a different type of post-viral brain pathology. Indeed, respiratory coronaviruses, prior to the CoV19 strain that cause human colds have been neuroinvasive, with viral RNA demonstrated in brain parenchyma as well as in myeloid microglia in culture. https://jvi.asm.org/content/74/19/8913 . It was  suspected that a subset of blood monocytes, not just neural olfactory spread, might be a conduit for CoV19 into brain with subsequent development of neuropsychiatric symptoms. A wide variety of neurologic complications and neuropathology have now been published, e.g., thromboemboli, infarction, radiologic changes consistent with an autoimmune encephalitis and even the presence of CoV19 in neurons. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086624/
 
https://pubmed.ncbi.nlm.nih.gov/32651556/
 
-2)Coronaviruses are but one group of many different viruses including influenza, rhinoviruses and adenoviruses that commonly infect the population. Infrequent lethal sudden deaths from uncharacterized winter "viral" infections on routine autopsy can show classical acute lymphocytic and myeloid infiltrates in an otherwise normal heart. Some of these might be due to a coronavirus strain. The recent collapse of healthy young athletes may be caused by CoV19 PBMC dissemination leading to vascular microthrombi in the heart and brain.
 
-3)The long-standing Yale-China relationship gave the study team the opportunity to attend a zoom meeting with doctors from Wuhan who shared their data and experience with in early in 2020. In one slide, a striking feature was the pathology of an interstitial pneumonia dominated by many large cells that were most consistent with a myeloid lineage. This further elicited the impetus to find if PBMC CoV19 infection could be a source of multi-organ spread.
 
The study team found over 300nt of CoV19 nucleocapsid RNA in PBMC indicating these cells can be a conduit for spread of the virus, or viral elements, to other organs
 
The use of NC primers adjacent to the 72bp NC RNA revealed strong evidence that long CoV19 NC sequences were present in patient PBMC. The two positive patient samples were validated by sequencing and completely matched 3 positive internal controls as well as the CoV19 NC sequences in the database.
 
Although the study team were limited to a small sample of PBMC, nominally ~6e4 cell RNA for 2-3 repeat replicate studies of each of two longer NC, it is clear that only a few select cells carry these sequences.
 
Importantly the fact that no one else has reported direct reproducible detection of CoV19 RNA sequences in PBMC indicates spread via these cells has not been widely considered even though they are a common route for the dissemination of many viruses to other organs.
 
Past coronavirus studies using mouse hepatitis virus (MHV), were previously presumed to spread by blood because lesions and antigens were distributed in a vascular pattern akin to the vascular pathology now reproducibly seen in COVID-19 patients. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086624/
 
After MHV intranasal infection, an infectious viremia with a significant titer was demonstrated by a colleague was in 1991. The virus was present in whole blood, plasma and buffy coat (white blood) cells 3 days post infection (4.1- 4.8 logs, LD50/ml, by a sensitive infant mouse titration assay). Moreover, by 5 days post infection this titer was maintained only in buffy coat cells. https://link.springer.com/article/10.1007/BF01321116
 
A biphasic clearance from plasma was also noted during the first 5 days post nasal inoculation, and importantly, the MHV viremia was intermittent. Many infectious agents are known to enter the bloodstream in spikes that coincide with intermittent fevers, and the study team suspects that patients with COVID-19 may not have a constant detectable level of viremia. The patients here were sampled only at one time point in disease and this could explain why only 20% of our patients were positive.
 
The MHV studies have relevance for the minor proportion of positives in CoV19 PBMC found here based on two factors:
 
 -First, our samples were taken 2-6 days after symptomatic admission, and these patients were probably infected at least 5 days before admission, a time when viremia may not be as high as during the asymptomatic phase.
 
-Second, the known clearance of MHV from blood during the initial infection could also lower the number of PBMC positives in patients evaluated here. In this context the study of plasma and PBMC at earlier phases of CoV19 exposure seems warranted. Such temporal evaluations could be useful diagnostically and also lead to antibody and other new strategies to prevent progressive spread to other organs. Assessing PBMC during later progressive disease with more severe organ dysfunction, or in more susceptible populations such as the elderly, may also be informative for understanding CoV19 viremic clearance or lack thereof. Notably, one patient who was 78 years old and her PBMC was taken 6 days after her symptoms had already developed along with a positive swab admission test.
 
The positive results here emphasize the importance of further studies to determine which PBMC cell type concentrates CoV19 sequences. The present small sample study shows a relatively low amount of CoV19 NC copies in total PBMC RNA, probably limited to a select cell type. Unfortunately,  the study team could not obtain more of the positive patient samples to assess combined CoV19 antigen and cell-type specific antibody tests. It is also important to determine if the subset of positive cells carry fully infectious CoV19, and not just the NC sequence, as for example by culture or animal inoculation. Electron microscopy for viral particles in selected cell types isolated by sorting, in addition to in-situ viral hybridization with antigenic cell-type specific markers, can be effectively performed on low numbers of subset cells.
 
The study team suspects that a circulating myeloid cell type probably carries CoV19 sequences or products, especially because these cells can take up residence in tissues and act as a latent source for infection and/or chronic immune stimulation. As antigen presenting cells, they are capable of stimulating immune responses that are detrimental to the host. Myeloid cells with CoV19 are also a likely viral conduit because of their known ability to attach and enter tissues via the vascular endothelium. New hamster and mouse transgenic angiotensin converting enzyme 2 receptor models of COVID-19 may be the most sensitive way to assay infectivity in such PBMC subsets. Identifying the positive cell subtype will also facilitate therapeutic approaches that specifically target these cells to prevent early dissemination of Cov19, in part or whole, to other organs.
 
The study team concluded that more studies are need to verify this findings and also to deal with the potential implications especially the huge staggering numbers of so called ‘recovered’ COVID-19 patients.
 
This is the third study in the last few days that is raising alarms and also causing a stir among the medical and research community. The first study to cause a stir was regarding findings that the SARS-CoV-2 RNA is able to integrate into the human host genome. https://www.thailandmedical.news/news/covid-19-news-harvard-and-mit-study-alarmingly-shows-that-sars-cov-2-rna-integrates-into-human-genome-with-varying-implications
 
The second was that the SARS-CoV-2 was able to cross the blood brain barrier. https://www.thailandmedical.news/news/new-covid-19-research-indicates-that-sars-cov-2-can-penetrate-the-blood-brain-barrier
 
There will be a few more interesting study findings coming out in the next 2 weeks.

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