A Comprehensive Analysis of the Covid-19 Crisis

[Patrick]

Dear Eddie, this is just gardening. We harvest what we have collectively sowed. Revolt is not the best way to sow something new in my opinion. I think we are already collectively sowing seeds of love and they will flourish with time. We just have to keep steady for the time being.

In practical terms, what would you suggest for someone like me, who is not going to get vaccinated but wants to travel again? 

Depending on what kind of travel you wish to do, I expect there will be destinations that does not ask for proof. Even here in Canada you will be able to enter without vaccination, but you will need to pay for and pass two negative PCR tests and quarantine for a couple days in a hotel while you wait for the second test results (one test before you fly and the second one is on arrival).

[the]

I am also interested in the 'revolution' timeline. I wrote this in another thread, edited and copied some of here.

https://www.bring4th.org/forums/showthre...#pid302154

Cobra , David Wilcock and many others has been talking about the 'military coup' timeline for years, although I am seeing it has less and less chance happening, but this it is an interesting timeline and worth investigation and preparation.

...
another thing, in terms of the 'revolution', after more and more people wake up, the 'revolution' becomes more and more peaceful, probably in the end we're in a timeline that the 'revolution' is so peaceful , it's more like a normal power transformation (transfer power back to the people), and no one really think that's a revolution.

but that all depends on peoples' consciousness level, the higher it goes, the peaceful it becomes. if the power transfer happens when not too many people reached high frequency, then it might becomes more violent and more casualty.
in other words, the timeline discussed in original video [Marina Jacobi - CYBORG VS HUMAN S4 E30 / New Info https://www.youtube.com/watch?v=o6D10XJoIL4] might be a 'worst case' scenario, but our end result might be better than that.

David Wilcock always present a 'military coup' timeline, positive military took all the power by force, and broadcast truth 24 hrs a day. I think that's just an emergency plan, but if that happens, many people's belief system will collapse, and they might be in depression or kill themselves. I am pretty sure the 'good guys' are also trying not to use that last approach.

but no matter which timeline we are going to experience in the end, we need to follow our heart all the way, don't because it's 'not peaceful', then suppress it and stop action. if it's time to act, then act upon it. many wanders come to this game to change the game, to ascend earth, not just watching the old 3D game.
just my humble opinion

[Diana]

Dear Eddie, this is just gardening. We harvest what we have collectively sowed. Revolt is not the best way to sow something new in my opinion. I think we are already collectively sowing seeds of love and they will flourish with time. We just have to keep steady for the time being.

In practical terms, what would you suggest for someone like me, who is not going to get vaccinated but wants to travel again? 

Depending on what kind of travel you wish to do, I expect there will be destinations that does not ask for proof. Even here in Canada you will be able to enter without vaccination, but you will need to pay for and pass two negative PCR tests and quarantine for a couple days in a hotel while you wait for the second test results (one test before you fly and the second one is on arrival).

Okay. Well, at least it isn't total restriction. I don't watch the news but I will have to start looking into it. I don't want to be isolated because I haven't been vaccinated. And I don't want to be forced into vaccination. 

[Margan]

The thing that has bugged me from the start is this assumption that you have to PROVE that you are healthy, and that everyone is potentially lethal and dangerous to everybody around them.
Normally I know when I am healthy and I know when I am sick. If one is not totally disconnected from the body this should be obvious.
And now we apparently need a test (which has been proven to be unreliable and create false positives) to tell us which is which....
just to confuse people and avoid them relying on their bodies and its signal.
And then, you have to automatically quarantine if going abroad unvaccinated -> solitary confinement or prison (although, I think at least you don't have to pay for your stay in prison, and afaik those quarantine hotels are quite expensive... ).
Yep the unwashed unvaccinated masses have to be punished methinks.

[Patrick]

Dear Eddie, this is just gardening. We harvest what we have collectively sowed. Revolt is not the best way to sow something new in my opinion. I think we are already collectively sowing seeds of love and they will flourish with time. We just have to keep steady for the time being.

In practical terms, what would you suggest for someone like me, who is not going to get vaccinated but wants to travel again? 

Depending on what kind of travel you wish to do, I expect there will be destinations that does not ask for proof. Even here in Canada you will be able to enter without vaccination, but you will need to pay for and pass two negative PCR tests and quarantine for a couple days in a hotel while you wait for the second test results (one test before you fly and the second one is on arrival).

Okay. Well, at least it isn't total restriction. I don't watch the news but I will have to start looking into it. I don't want to be isolated because I haven't been vaccinated. And I don't want to be forced into vaccination. 

I don't think it will be possible to truly completely enforce vaccination for traveling, because some people simply cannot be vaccinated for medical reasons. So there will have to be alternatives, like the ones Canada has put in place. But I think they will make it a hassle as much as possible so that it encourages people to just get vaccinated and be done with it. That is pretty much why I got vaccinated. I don't care about the protection and I don't fear that virus. I just wished to have an easier time travelling. (But I also do not fear those vaccines one iota)

[Eddie]

Here's another article, linking the COVID panic to immense (multi-trillion $) financial fraud:

https://gizadeathstar.com/2021/07/a-plan...old-heist/

[tadeus]

New peer-reviewed study confirms: NO VIRUS needed to infect with SARS-CoV-2!

SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2

[Eddie]

Moderna had Covid-19 Shot Ready Before Pandemic Was Announced

Joseph Farrell's commentary on the above:

Operation WarpSpeed, or Foreknowledge?

[David_1]

Suppose you think you are doing what is best.
You follow the directions given to you.
What happens when you figure out they lied?
So sad.
https://tinyurl.com/6ems6drm

[tadeus]

WHO issues new recommendations on human genome editing for the advancement of public health

[Eddie]

Genetically Engineered "Magneto" Protein Remotely Controls Brain and Behavior

[Eddie]

COVID Surge In Most-Vaxxed Countries:

https://www.zerohedge.com/covid-19/worry...surge-most

(This appears to be translated from some language other than English, and the translation is bad, but the article gets the point across.)

Another article:

CDC Quietly Deletes 600 Covid-vaccine Deaths from Its Web Site in One Day

[David_1]

Australia has fallen into demonic enslavement.
Will your country be next?
https://www.youtube.com/watch?v=H8KScb7XjzE

[pat19989]

Australia has fallen into demonic enslavement.
Will your country be next?
https://www.youtube.com/watch?v=H8KScb7XjzE

one can only hope.

demonic enslavement sounds like efficient catalyst!

[Diana]

Australia has fallen into demonic enslavement.
Will your country be next?
https://www.youtube.com/watch?v=H8KScb7XjzE

I don't know about demonic, but it is certainly enslavement. 

[Patrick]

Today, even the "opposition" is calling for our provincial prime minister to implement a vaccine passport right away.

[Diana]

Today, even the "opposition" is calling for our provincial prime minister to implement a vaccine passport right away.

By the opposition, who are you referring to?

[Patrick]

Today, even the "opposition" is calling for our provincial prime minister to implement a vaccine passport right away.

By the opposition, who are you referring to?

The opposition leader in government.

https://montrealgazette.com/news/quebec/...-passports

[Patrick]

I just don't get this push.

We are nearing 85% of the population with a first dose and 72% with 2 doses. That is astoundingly good in my opinion. They should be more than happy with that response. If we add the 10% that got Covid, we can say we are at Herd Immunity and beyond.

At this point they just want us to go through this societal catalyst of "freedom passports".

Just naming it that is them laughing in our faces.

[Nikki]

I have read all of the posts in this thread and must say there is so much love and wanting to help others. I will leave right now but will be back to post. Meanwhile, I leave with this poem for all that is shared in love.

“Out beyond ideas of wrongdoing and rightdoing,
there is a field. I’ll meet you there.
When the soul lies down in that grass,
the world is too full to talk about.
Ideas, language, even the phrase “each other”
doesn’t make any sense.
The breeze at dawn has secrets to tell you.
Don’t go back to sleep.
You must ask for what you really want.
Don’t go back to sleep.
People are going back and forth across the doorsill
where the two worlds touch.
The door is round and open.
Don’t go back to sleep.” Rumi

[Patrick]

Just the first 20 seconds of this vid says it all...

[Relax]

Coronavirus biology and replication: implications for SARS-CoV-2

   Philip V’kovski, Annika Kratzel, Silvio Steiner, Hanspeter Stalder & Volker Thiel

Nature Reviews Microbiology volume 19, pages 155–170 (2021) Cite this article

   277k Accesses 401 Altmetric Metrics details [NB: Published: 28 October 2020]

Abstract

The SARS-CoV-2 pandemic and its unprecedented global societal and economic disruptive impact has marked the third zoonotic introduction of a highly pathogenic coronavirus into the human population. Although the previous coronavirus SARS-CoV and MERS-CoV epidemics raised awareness of the need for clinically available therapeutic or preventive interventions, to date, no treatments with proven efficacy are available. The development of effective intervention strategies relies on the knowledge of molecular and cellular mechanisms of coronavirus infections, which highlights the significance of studying virus–host interactions at the molecular level to identify targets for antiviral intervention and to elucidate critical viral and host determinants that are decisive for the development of severe disease. In this Review, we summarize the first discoveries that shape our current understanding of SARS-CoV-2 infection throughout the intracellular viral life cycle and relate that to our knowledge of coronavirus biology. The elucidation of similarities and differences between SARS-CoV-2 and other coronaviruses will support future preparedness and strategies to combat coronavirus infections.

Introduction

Coronaviruses (CoVs) are a highly diverse family of enveloped positive-sense single-stranded RNA viruses. They infect humans, other mammals and avian species, including livestock and companion animals, and are therefore not only a challenge for public health but also a veterinary and economic concern. Within the order of Nidovirales and the suborder of Coronavirineae lies the family Coronaviridae. The latter is further specified into the subfamily of Orthocoronavirinae, which consists of four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. Whereas alphacoronaviruses and betacoronaviruses exclusively infect mammalian species, gammacoronaviruses and deltacoronaviruses have a wider host range that includes avian species. Human and animal coronavirus infections mainly result in respiratory and enteric diseases1,2.

Human coronaviruses, such as HCoV-229E and HCoV-OC43, have long been known to circulate in the population and they, together with the more recently identified HCoV-NL63 and HCoV-HKU1, cause seasonal and usually mild respiratory tract infections associated with symptoms of the ‘common cold’. In strong contrast, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2, which have emerged in the human population over the past 20 years, are highly pathogenic. By infecting bronchial epithelial cells, pneumocytes and upper respiratory tract cells in humans, SARS-CoV, MERS-CoV and SARS-CoV-2 infections can develop into severe, life-threatening respiratory pathologies and lung injuries for which no specific prophylactic or therapeutic treatment has been approved to date.

The initial steps of coronavirus infection involve the specific binding of the coronavirus spike (S) protein to the cellular entry receptors, which have been identified for several coronaviruses and include human aminopeptidase N (APN; HCoV-229E), angiotensin-converting enzyme 2 (ACE2; HCoV-NL63, SARS-CoV and SARS-CoV-2) and dipeptidyl peptidase 4 (DPP4; MERS-CoV). The expression and tissue distribution of entry receptors consequently influence viral tropism and pathogenicity. During the intracellular life cycle (Fig. 1), coronaviruses express and replicate their genomic RNA to produce full-length copies that are incorporated into newly produced viral particles. Coronaviruses possess remarkably large RNA genomes flanked by 5′ and 3′ untranslated regions that contain cis-acting secondary RNA structures essential for RNA synthesis. At the 5′ end, the genomic RNA features two large open reading frames (ORFs; ORF1a and ORF1b) that occupy two-thirds of the capped and polyadenylated genome. ORF1a and ORF1b encode 15–16 non-structural proteins (nsp), of which 15 compose the viral replication and transcription complex (RTC) that includes, amongst others, RNA-processing and RNA-modifying enzymes and an RNA proofreading function necessary for maintaining the integrity of the >30 kb coronavirus genome3. ORFs that encode structural proteins and interspersed ORFs that encode accessory proteins are transcribed from the 3′ one-third of the genome to form a nested set of subgenomic mRNAs (sg mRNAs). Coronavirus accessory proteins are highly variable sets of virus-specific proteins that display limited conservation even within individual species but they are principally thought to contribute to modulating host responses to infection and are determinants of viral pathogenicity4,5. Nevertheless, the molecular functions of many accessory proteins remain largely unknown owing to the lack of homologies to accessory proteins of other coronaviruses or to other known proteins6.

[data table image]

Despite the previous public health emergencies caused by the SARS-CoV and MERS-CoV outbreaks and the impact of the ongoing SARS-CoV-2 pandemic on society and human health, intervention strategies to combat coronavirus infections are only in their early stages and await proof of clinical efficacy. Their development intimately relies on the deepened understanding of basic mechanisms of coronavirus gene functions as well as of the molecular interactions with host factors. Since the discovery of the first coronavirus (avian infectious bronchitis virus) in the 1930s7 and the discovery of the first human coronaviruses (HCoV-229E and HCoV-OC43) in the 1960s8,9, the coronavirus research field has made substantial progress in understanding the basic principles of coronavirus replication and pathogenesis (Box 1). This advancement was accelerated after the emergence of SARS-CoV in 2002 and MERS-CoV in 2012 and has broadened our view on coronaviruses as zoonotic pathogens that can severely affect human health. Moreover, the unprecedented speed and technical progress of coronavirus research that has become evident in a few months after the appearance of SARS-CoV-2 at the end of 2019 has led to a rapidly growing understanding of this newly emerging pathogen and of its associated disease, COVID-19. In this Review, we discuss key aspects of coronavirus biology and their implications for SARS-CoV-2 infections as well as the treatment and prevention strategies.

[TBC below]

https://www.nature.com/articles/s41579-020-00468-6

[Relax]

Entry of coronaviruses

Coronavirus S proteins are homotrimeric class I fusion glycoproteins that are divided into two functionally distinct parts (S1 and S2) (Fig. 2). The surface-exposed S1 contains the receptor-binding domain (RBD) that specifically engages the host cell receptor, thereby determining virus cell tropism and pathogenicity. The transmembrane S2 domain contains heptad repeat regions and the fusion peptide, which mediate the fusion of viral and cellular membranes upon extensive conformational rearrangements10,11,12. Shortly after the 2002–2003 SARS-CoV outbreak, ACE2 was identified as the functional receptor that enables infection by SARS-CoV13. The high genomic and structural homology between the S proteins of SARS-CoV and SARS-CoV-2 (76% amino acid identity) supported the identification of ACE2 as the cell-surface receptor for SARS-CoV-2 (refs12,14,15,16). Remarkably, essential SARS-CoV contact residues that interact with ACE2 were highly conserved in SARS-CoV-2 as well as in members of the species Severe acute respiratory syndrome-related coronavirus that use ACE2 or have similar amino acid side chain properties14,15,17,18,19. These data were corroborated by the atomic resolution of the interface between the SARS-CoV-2 S protein and ACE2 (refs16,19,20,21). By contrast, the bat Severe acute respiratory syndrome-related coronavirus RaTG13 S sequence (93.1% nucleotide identity to SARS-CoV-2) shows conservation of only one out of six amino acids directly involved in ACE2 binding, even though, based on the entire genomic sequence, RaTG13 is the closest relative of SARS-CoV-2 known to date (96.2%)14 (Box 2).

Viral gene expression and RNA synthesis
Genome translation

The release of the coronavirus genome into the host cell cytoplasm upon entry marks the onset of a complex programme of viral gene expression, which is highly regulated in space and time. The translation of ORF1a and ORF1b from the genomic RNA produces two polyproteins, pp1a and pp1ab, respectively. The latter results from a programmed –1 ribosomal frameshift at the short overlap of ORF1a and ORF1b4. Ribosome profiling revealed that the efficiency of the frameshift between ORF1a and ORF1b lies between 45% and 70% in the case of SARS-CoV-2 (ref.60), similar to that measured for mouse hepatitis virus (MHV)61. This determines the stoichiometry between pp1a and pp1ab, with pp1a being approximately 1.4–2.2 times more expressed than pp1ab60. Sixteen non-structural proteins are co-translationally and post-translationally released from pp1a (nsp1–11) and pp1ab (nsp1–10, nsp12–16) upon proteolytic cleavage by two cysteine proteases that are located within nsp3 (papain-like protease; PLpro) and nsp5 (chymotrypsin-like protease) (Fig. 3). The protease residing in nsp5 is frequently referred to as 3C-like protease (3CLpro), because of its similarities to the picornaviral 3C protease, or as main protease (Mpro), because it is responsible for proteolytic processing of the majority of polyprotein cleavage sites. Proteolytic release of nsp1 is known to occur rapidly62, which enables nsp1 to target the host cell translation machinery63,64,65. Nsp2–16 compose the viral RTC and are targeted to defined subcellular locations where interactions with host cell factors determine the course of the replication cycle66,67,68. Nsp2–11 are believed to provide the necessary supporting functions to accommodate the viral RTC, such as modulating intracellular membranes, host immune evasion and providing cofactors for replication, whereas nsp12–16 contain the core enzymatic functions involved in RNA synthesis, RNA proofreading and RNA modification4,67. RNA synthesis is performed by the nsp12 RNA-dependent RNA polymerase (RdRP) and its two cofactors nsp7 and nsp8, the latter with proposed primase or 3′-terminal adenylyltransferase activity4,67,69,70. Notably, nsp14 provides a 3′–5′ exonuclease activity that assists RNA synthesis with a unique RNA proofreading function71. The coronavirus capping machinery, which is not yet fully elucidated, is composed of nsp10, which functions as a cofactor, nsp13, which provides the RNA 5′-triphosphatase activity, and nsp14 and nsp16, which perform the functions of N7-methyltransferase and 2′-O-methyltransferase, respectively67,72,73,74. Notably, one key enzyme typically involved in the formation of the 5′ cap structure, the guanylyltransferase, has not yet been identified in coronaviruses.

The establishment of the viral RTC is crucial for virus replication and thus a promising target for antivirals against SARS-CoV-2. One such target is Mpro, which resides in nsp5. Mpro releases the majority of nsps from the polyproteins and is essential for the viral life cycle. Furthermore, as Mpro is very sequence specific, compounds that structurally mimic those cleavage sites can specifically target the viral protease with little or no impact on host cellular proteases75,76,77. Based on structural analysis of the protein, multiple research groups have successfully developed lead compounds that block Mpro function in cell culture assays, thus providing frameworks that could aid in rapid drug discovery75,77.
RNA synthesis

Viral genomic replication is initiated by the synthesis of full-length negative-sense genomic copies, which function as templates for the generation of new positive-sense genomic RNA. These newly synthesized genomes are used for translation to generate more nsps and RTCs or are packaged into new virions. A hallmark of coronaviruses and most members of the order Nidovirales is the discontinuous viral transcription process, first proposed by Sawicki and Sawicki78, that produces a set of nested 3′ and 5′ co-terminal subgenomic RNAs (sgRNAs)78,79 (Fig. 4). During negative-strand RNA synthesis, the RTC interrupts transcription following the encounter of transcription regulatory sequences (TRSs) that are located upstream to most ORFs in the 3′ one-third of the viral genome. At these TRS elements, also called TRS ‘body’, the synthesis of the negative-strand RNA stops and is re-initiated at the TRS adjacent to a leader sequence (TRS-L) located at about 70 nucleotides from the 5′ end of the genome78,79,80,81,82. This discontinuous step of coronavirus RNA synthesis involves the interaction between complementary TRSs of the nascent negative strand RNA (negative-sense TRS body) and the positive strand genomic RNA (positive-sense TRS-L). Upon re-initiation of RNA synthesis at the TRS-L region, a negative strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs. The discontinuous step of negative strand RNA synthesis results in the production of a set of negative-strand sgRNAs that are then used as templates to synthesize a characteristic nested set of positive-sense sg mRNAs that are translated into structural and accessory proteins. Although the coronavirus sg mRNAs are structurally polycistronic, it is assumed that they are functionally monocistronic and that only the first ORF at the 5′ end, which is absent in the next smaller sgRNA, is translated from each sgRNA78,81.

The TRS elements for SARS-CoV-2 have already been determined by RNA sequencing analyses of viral RNAs80,83. Like for SARS-CoV, the consensus TRS core of SARS-CoV-2 is 5′-ACGAAC-3′ and eight sg mRNAs have been shown to be produced in SARS-CoV-2-infected cells (sg mRNAs 2–9). In addition to canonical sgRNAs, recent reports also determined the existence of numerous non-canonical RNA products of discontinuous transcription, including fusions of the 5′ leader sequence to unexpected 3′ sites, TRS-L independent long-distance fusions, and local fusions resulting in small deletions mainly in the structural and accessory genes60,80. However, it remains to be determined whether all of these non-canonical sgRNAs truly arise by discontinuous transcription or whether they represent RNAs that result from recombination. Nevertheless, similar findings were previously reported for other coronaviruses, including MHV61 and HCoV-229E81, which indicates an enhanced coding potential for coronaviruses80. Overall, these unexpected fusion events may drive coronavirus evolution through variant generation, and novel ORFs could encode additional accessory proteins that are involved in either viral replication or modulation of the host immune response60,80.

The RdRP residing in nsp12 is the centrepiece of the coronavirus RTC and has been suggested as a promising drug target as it is a crucial enzyme in the virus life cycle both for replication of the viral genome but also for transcription of sgRNAs. The structure of the SARS-CoV-2 RdRP nsp12 and its cofactors nsp7 and nsp8 has been elucidated and shows a high degree of conservation to the SARS-CoV structure69,84,85. The amino acid sequence of the SARS-CoV and SARS-CoV-2 RdRPs show a >95% similarity with most changes located in the nidovirus RdRP-associated nucleotidyltransferase domain, which, despite being a genetic marker of Nidovirales, has yet to be functionally elucidated69. The structural similarities of the RdRP active site, including conserved key amino acid residues, with other positive-sense RNA viruses suggest the possibility to repurpose known drugs that are effective against other RNA viruses69. One of the most promising candidates is the phosphoramidate remdesivir (RDV), which, in its triphosphate form, acts as a substrate for viral RdRPs and competes with ATP86. RDV has shown potential as an antiviral agent against a broad range of RNA viruses, including Filoviridae (for example, Ebola virus), Paramyxoviridae (for example, Nipah virus) and Pneumoviridae (for example, respiratory syncytial virus) as well as other coronaviruses, including SARS-CoV and MERS-CoV86,87. The RdRP of SARS-CoV-2 selectively incorporates RDV over ATP, which subsequently results in a delayed-chain termination86,88. In contrast to classic nucleoside analogues that lead to immediate termination of the synthesis reaction after incorporation, the RdRP continues for three nucleotides after RDV has been incorporated before chain termination. Nucleotide analogues like RDV may have limited efficacy owing to the proofreading function of the exonuclease domain contained in nsp14 (ExoN)89. The corrective function that is exerted by ExoN is not only responsible for maintaining the stability of the coronavirus genome but also enables the excision of erroneous mutagenic nucleotides71,89. The mode of action observed for RDV might be an explanation for its increased efficiency over other nucleoside analogues as the delayed-chain termination could lead to improved evasion from the proofreading function of nsp14. The current model suggests steric hindrance as a likely reason for termination, disturbing the positioning of the RNA and thus hampering the translocation to the next position86,88. RDV was shown to reduce virus replication of SARS-CoV-2 in vitro90 and was demonstrated to restrict clinical symptoms of SARS-CoV-2 in rhesus macaques upon early pre-symptomatic treatment91. However, a recent randomized, double-blind, placebo-controlled clinical trial in humans with severe COVID-19 showed limited clinical efficacy of RDV treatment92 and further studies will be necessary. Another promising candidate is the purine analogue favipiravir (FPV), which has been shown to effectively target multiple RNA viruses93. Although the mechanism of action is not yet completely understood, a recent study of the in vitro mechanism of FPV suggested a combination of chain termination, slowed RNA synthesis and lethal mutagenesis as the mode of action against SARS-CoV-2, which indicates that FPV might be used to effectively restrict viral replication93. Indeed, results of an experimental pilot study showed that using FPV as treatment against COVID-19 led to increased recovery and faster viral clearance times in treated patients compared to control treatments94. Clinical studies with both RDV and FPV are currently ongoing and will establish whether these compounds are effective antivirals to treat coronavirus infections93.

[TBC below]

https://www.nature.com/articles/s41579-020-00468-6

[Relax]

Expression of structural and accessory proteins

The ORFs encoding the structural proteins (that is, S protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein) are located in the 3′ one-third of coronavirus genomes. Interspersed between these ORFs are the ORFs encoding for so-called accessory proteins. The structural proteins of SARS-CoV-2 have not yet been assessed in terms of their role in virus assembly and budding. In general, coronavirus structural proteins assemble and assist in the budding of new virions at the endoplasmic reticulum (ER)-to-Golgi compartment that are suggested to exit the infected cell by exocytosis95,96,97. However, recent evidence shows that betacoronaviruses, including MHV and SARS-CoV-2, rather egress infected cells via the lysosomal trafficking pathway98. During this process, viral interference with lysosomal acidification, lysosomal enzyme activity and antigen presentation was demonstrated.

At least five ORFs encoding accessory genes have been reported for SARS-CoV-2: ORF3a, ORF6, ORF7a, ORF7b and ORF8 (GenBank entry NC_045512.2) as well as potentially ORF3b99 and ORF9b100, the latter of which is probably expressed as a result of leaky scanning of the sgRNA of the nucleocapsid protein80,99,101. In addition, ORF10 has been postulated to be located downstream of the N gene. However, not all of these ORFs have been experimentally verified yet and the exact number of accessory genes of SARS-CoV-2 is still debated80,102. For example, in the case of ORF10, recent sequencing data questioned whether ORF10 is actually expressed, as the corresponding sgRNA could only be detected once in the entire dataset80. Furthermore, using proteomics approaches, the ORF10 protein has not been found in infected cells100,102, whereas ribosome profiling data suggested that ORF10 may be translated60.

The accessory genes display a high variability among coronavirus groups and usually show no sequence similarity with other viral and cellular proteins. Although they are not required for virus replication in cell culture4,5, they are, to some extent, conserved within the respective virus species and are suspected to have important roles in the natural host. Indeed, in the case of SARS-CoV, it was shown that at least ORF3b, ORF6 and ORF9b function as interferon antagonists6,102,103,104. There are some notable differences between the accessory genes of SARS-CoV-2 and SARS-CoV, with the latter having a total of eight described accessory genes (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, ORF8b and ORF9b). In SARS-CoV-2, ORF3b contains a premature stop codon and is thus substantially shorter than the SARS-CoV variant. Although there are indications that ORF3b could exhibit its interferon antagonistic function also in a truncated form99, it has not yet been found to be expressed at the protein level in virus-infected cells100,102. SARS-CoV-2 ORF8 shows an especially low homology to SARS-CoV ORF8. The coding sequence of SARS-CoV ORF8 went through a gradual deletion over the course of the SARS-CoV epidemic. Whereas the early isolates from human patients contained a full-length ORF8, a deletion of 29 nucleotides was observed in all SARS-CoV strains during the middle-to-late stages. This deletion caused the split of one full-length ORF8 into two truncated gene products, ORF8a and ORF8b. Furthermore, less frequent deletion events were also observed, including an 82-nucleotide deletion and a 415-nucleotide deletion, which led to a complete loss of ORF8 (refs105,106), suggesting a possible benefit of SARS-CoV ORF8 deletions in vivo. Notably, however, reconstitution of SARS-CoV ORF8 by reverse genetics was associated with slightly increased fitness in cell culture106. Recently, SARS-CoV-2 ORF8 was reported to bind to major histocompatibility complex and mediate its degradation in cell culture107. This indicates that SARS-CoV-2 ORF8 might mediate a form of immune evasion, which is not the case for the split SARS-CoV ORF8a or ORF8b107. Interestingly, a mutant SARS-CoV-2 strain found in Singapore displayed a deletion of 382 nucleotides in the region of ORF8, indeed spanning most of the ORF and the adjacent the TRS. This may indicate a tendency towards host adaption and decreased pathogenicity108 or, alternatively, that the ORF8 protein is dispensable in humans, whereas it is required in the natural host.

Replication compartments

Primary interactions between nsps and host cell factors during the early coronavirus replication cycle initiate the biogenesis of replication organelles66,109,110. Although mechanisms underlying replication organelle formation are not fully understood, the concerted role of the membrane-spanning nsp3, nsp4 and nsp6 has been implicated in diverting host endomembranes into replication organelles111,112,113. Detailed electron microscopy investigations have described the phenotypic appearance and extent of membrane modifications induced by coronaviruses to accommodate viral replication. Coronavirus infection, like many other positive-sense RNA viruses, manifests in the generation of ER-derived and interconnected perinuclear double-membrane structures such as double-membrane vesicles (DMVs), convoluted membranes and the recently discovered double-membrane spherules112,114,115,116. Interestingly, these structures are highly dynamic and develop during the viral life cycle114,117. Even though replicase subunits — notably SARS-CoV nsp3, nsp5 and nsp8 — have been shown to be anchored on convoluted membranes, to date, the specific location of viral RNA synthesis remains the most intriguing unanswered question114,117. Double-stranded RNA (dsRNA), commonly considered as viral replication intermediates, segregates into the DMV interior97,114,118. Consistently, viral RNA synthesis was shown to occur within DMVs by using metabolic labelling of newly synthesized viral RNA in the context of SARS-CoV, MERS-CoV and infectious bronchitis virus infections116. Although, until recently, no openings towards the cytosol have been observed97,114, molecular pores involving nsp3 were demonstrated to span DMVs in MHV-infected cells118. These newly identified structures, which were also observed in SARS-CoV-2-infected cells, provide a connection between the dsRNA-containing DMV interior and the cytosol, thereby hypothetically rendering newly synthesized viral RNAs available for translation and encapsidation into nascent virions118. They also provide new opportunities to experimentally address the origin, fate and trafficking routes of viral RNAs contained in DMVs.

Replication organelles are a conserved and characteristic feature of coronavirus replication and, consistent with suggested roles of rewired intracellular membranes in the context of other positive-sense RNA virus infections, they provide a propitious niche with adequate concentrations of macromolecules necessary for RNA synthesis while preventing the exposure of viral replication intermediates to cytosolic innate immune sensors95,119. The functional dissection of coronavirus replication organelles has proven challenging as their contributions to viral fitness and pathogenesis are indistinguishable from functions provided by enzymes of the RTC, which are anchored on the membranes of the replication organelle120,121,122. Nevertheless, recent studies revealed the overall composition of the coronavirus RTC, with nsp2–nsp16 and the nucleocapsid protein comprising the viral components68,123. Moreover, several genetic and proteomic screening approaches aimed at deciphering essential coronavirus–host interactions and the RTC microenvironment identified supportive roles of the ER and the early secretory system as well as related vesicular trafficking pathways for efficient replication68,124,125,126 and provided a comprehensive list of cellular proteins that are in close proximity to the coronaviral RTC68,127,128,129. Collectively, these studies, in combination with advanced electron microscopy, provide ground for future studies to dissect the microarchitecture of the coronaviral RTC in relation to remodelled ER-derived membranes and to functionally link those structures to processes taking place in close proximity to the RTC such as translation, replication and transcription of viral RNA.
[TBC below]

https://www.nature.com/articles/s41579-020-00468-6

[Relax]

Virus–host interactions and host response

A successful intracellular coronavirus life cycle invariably relies on critical molecular interactions with host proteins that are repurposed to support the requirements of the virus. This includes host factors required for virus entry (such as the entry receptor and host cell proteases), factors required for viral RNA synthesis and virus assembly (such as ER and Golgi components and associated vesicular trafficking pathways) and factors required for the translation of viral mRNAs (such as critical translational initiation factors)68,124,125,126,127,128,129.

A first systematic expression study of SARS-CoV-2 proteins and subsequent affinity purification followed by mass spectrometry identified more than 300 potential coronavirus–host protein interactions. Although outside the context of a SARS-CoV-2 infection, interactors of individually overexpressed SARS-CoV-2 proteins uncovered several cellular processes reminiscent of those of other coronaviruses that are likely to also be involved in the SARS-CoV-2 life cycle130. Importantly, 69 compounds, either FDA approved or at different stages of clinical development, that target putative SARS-CoV-2 protein interactors were foregrounded, a subset of which efficiently prevented SARS-CoV-2 replication in vitro. These systematic screening approaches of large compound libraries that target host proteins provide means of rapidly identifying antiviral (repurposed) drugs and accelerated clinical availability131. However, a detailed functional characterization of conserved host pathways that promote coronavirus replication will guide the development of efficacious targeted therapeutics against coronavirus infections.

In addition, coronaviruses efficiently evade innate immune responses. Virus–host interactions in this context are multifaceted and include strategies to hide viral pathogen-associated molecular patterns, such as replication intermediates (dsRNA), that may be sensed by cytosolic pattern recognition receptors132,133. DMVs have been proposed to shield dsRNA and sites of viral RNA synthesis; however, experimental proof supporting this idea has not yet been obtained. The coronaviral RTC also contributes to innate immune evasion through several nsp-encoded functions. These include PLpro-mediated deubiquitylation activity134,135, de-ADP-ribosylation by nsp3-encoded macro domains136, RNA-modifying enzymatic activities such as 5′-cap N7-methylation and 2′-O-methylation (nsp14 and nsp16, respectively)74,137,138, and exonuclease139 and endoribonuclease140,141 activities (nsp14 and nsp15, respectively). Although these mechanisms have been elucidated in considerable detail for several prototype coronaviruses, data for SARS-CoV-2 are not yet available.

Besides the well-conserved functions residing in the nsps that comprise the RTC, additional mechanisms to counteract innate immune responses are known for coronaviruses. For example, nsp1 is rapidly proteolytically released from pp1a and pp1ab and affects cellular translation in the cytoplasm to favour viral mRNAs over cellular mRNA, and thereby also decreases the expression of type I and III interferons and of other host proteins of the innate immune response. Indeed, a first structural and functional analysis of SARS-CoV-2 nsp1 showed binding of nsp1 to ribosomes and nsp1-mediated impairment of translation64. Furthermore, several coronavirus accessory proteins are known to affect innate immune responses, most prominently MHV NS2 and MERS-CoV ORF4b proteins, that have 2′,5′-phosphodiesterase activity to antagonize the OAS–RNase L pathway142. Although this activity is not predicted for any accessory protein of SARS-CoV or SARS-CoV-2, the ORF3b, ORF6 and N proteins of SARS-CoV have been shown to interfere at multiple levels of the cellular interferon signalling pathway, thereby efficiently inhibiting innate immune responses103. Interestingly, an initial report recently suggested a similar role of SARS-CoV-2 ORF3b as an effective interferon antagonist99. Although this property remains to be demonstrated in the context of viral infection, these results suggest that SARS-CoV-2 shares some preserved accessory protein activities with SARS-CoV that interfere with antiviral host responses.
Coronavirus biology and COVID-19

Our knowledge on SARS-CoV-2 replication, gene function and host interactions is accumulating at unprecedented speed and it will be important to link those findings to the disease induced by SARS-CoV-2 infection, COVID-19. Thus, there is a need to establish experimental systems, such as representative animal models to study the transmission and pathogenicity of SARS-CoV-2, primary airway epithelial cultures and organoids to study SARS-CoV-2 replication and host responses to infection in relevant cell types, and reverse genetics systems to study the specific gene functions of SARS-CoV-2 (Table 1). These tools will be instrumental to understanding how the molecular biology of SARS-CoV-2 affects the development of COVID-19.

As we currently understand, SARS and COVID-19 are a consequence of virus-encoded functions and delayed interferon responses and, in severe cases, they are associated with dysregulated immune responses and immunopathologies143,144. Indeed, rapid and uncontrolled viral replication of SARS-CoV has been demonstrated to evade the host innate immune activation during its initial steps. As a consequence, the increase in aberrant pro-inflammatory responses and immune cell infiltration in the lungs provoke tissue damage and contribute to the clinical manifestation of SARS145.

Consistently, host responses, such as cytokine expression, that are known to drive inflammation and immunopathologies have been assessed in studies that revealed that SARS-CoV-2 considerably affects the transcriptional landscape of infected cells by inducing inflammatory cytokine and chemokine signatures38,146,147. Although interferon responses have been shown to potently impair SARS-CoV-2 replication, only moderate induction of type I interferon, type II interferon and interferon-stimulated genes was reported38,147.

Together, these effects may translate into strong and dysregulated pro-inflammatory responses, while cells display low innate antiviral defence activation as revealed by single-cell transcriptomic studies of nasopharyngeal and bronchial patient samples38,146,148,149. In severe COVID-19 cases, as opposed to mild cases, aberrant recruitment of inflammatory macrophages and infiltration of T lymphocytes, including cytotoxic T cells, as well as of neutrophils have been measured in the lung146,149. The accumulating evidence of dysregulated pro-inflammatory responses during SARS-CoV-2 infections has led to the use of immune modulators to inhibit hyperactivated pathogenic immune responses143,144,150,151

Conclusions

In contrast to the SARS-CoV epidemic of almost 20 years ago, improved technologies, such as transcriptomics, proteomics, single-cell RNA sequencing, global single-cell profiling of patient samples, advanced primary 3D cell cultures and rapid reverse genetics, have been valuable tools to understand and tackle SARS-CoV-2 infections. Furthermore, several existing animal models initially established for SARS-CoV are applicable to study SARS-CoV-2 and will help to identify the critical viral and host factors that impact on COVID-19. We need to understand why SARS-CoV-2, in contrast to SARS-CoV, is replicating so efficiently in the upper respiratory tract and which viral and host determinants are decisive on whether COVID-19 patients will develop mild or severe disease152,153,154. Finally, we need to put the first encouraging studies on SARS-CoV-2 into the context of coronavirus biology to develop efficacious strategies to treat COVID-19 and to develop urgently needed vaccines.

References

   1. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 5, 536–544 (2020). Most recent update of the coronavirus taxonomy by the International Committee on Taxonomy of Viruses after the emergence of SARS-CoV-2. Coined the virus name SARS-CoV-2.
   
   2. Corman, V. M., Muth, D., Niemeyer, D. & Drosten, C. Hosts and sources of endemic human coronaviruses. Adv. Virus Res. 100, 163–188 (2018).
   
   3. Gorbalenya, A. E., Enjuanes, L., Ziebuhr, J. & Snijder, E. J. Nidovirales: evolving the largest RNA virus genome. Virus Res. 117, 17–37 (2006).
   
   4. Perlman, S. & Netland, J. Coronaviruses post-SARS: update on replication and pathogenesis. Nat. Rev. Microbiol. 7, 439–450 (2009).

   5. Masters, P. S. The molecular biology of coronaviruses. Adv. Virus Res. 65, 193–292 (2006).

   6. Liu, D. X., Fung, T. S., Chong, K. K. L., Shukla, A. & Hilgenfeld, R. Accessory proteins of SARS-CoV and other coronaviruses. Antivir. Res. 109, 97–109 (2014).

   7. Schalk, A. & Hawn, M. C. An apparently new respiratory disease of baby chicks. J. Am. Vet. Med. Assoc. 78, 413–423 (1931).

   8. Hamre, D. & Procknow, J. J. A new virus isolated from the human respiratory tract. Proc. Soc. Exp. Biol. Med. 121, 190–193 (1966).

   9. McIntosh, K., Dees, J. H., Becker, W. B., Kapikian, A. Z. & Chanock, R. M. Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease. Proc. Natl Acad. Sci. USA 57, 933–940 (1967).

  10. Tortorici, M. A. & Veesler, D. Structural insights into coronavirus entry. Adv. Virus Res. 105, 93–116 (2019).

  11. Li, F. Structure, function, and evolution of coronavirus spike proteins. Annu. Rev. Virol. 3, 237–261 (2016).

  12. Letko, M., Marzi, A. & Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol. 5, 562–569 (2020). First functional assessment of the interaction of the SARS-CoV-2 Spike protein receptor binding domain with the cellular receptor ACE2.

   13. Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454 (2003).

   14. Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020).

   15. Hoffmann, M. et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280 (2020). Functional assessment of SARS-CoV-2 entry into host cells highlighting the importance of the ACE2 receptor and the cellular protease TMPRSS2 as entry factors.

   16. Walls, A. C. et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 181, 281–292 (2020). Recommended resource describing key aspects of SARS-CoV-2 Spike features.

   17. Li, F., Li, W., Farzan, M. & Harrison, S. C. Structural biology: structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864–1868 (2005).

   18. Li, W. et al. Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J. 24, 1634–1643 (2005)...........................
   
   ...................33. Hamming, I. et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J. Pathol. 203, 631–637 (2004).

   34.
Shieh, W. J. et al. Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan. Hum. Pathol. 36, 303–309 (2005)....................

................ 94. Cai, Q. et al. Experimental treatment with favipiravir for COVID-19: an open-label control study. Engineering https://doi.org/10.1016/j.eng.2020.03.007 (2020).

   95. Stertz, S. et al. The intracellular sites of early replication and budding of SARS-coronavirus. Virology 361, 304–315 (2007)...............

  .................[NB: see link for another 105 Scientific References]

[NB: Published: 28 October 2020
Coronavirus biology and replication: implications for SARS-CoV-2

   Philip V’kovski, Annika Kratzel, Silvio Steiner, Hanspeter Stalder & Volker Thiel

Glossary

Nested set

   Refers to the nested set of coronavirus 5′-coterminal and 3′-coterminal RNAs. Most nidoviruses (Nidus, nest, latin) share this mechanism of transcription and are grouped into the order Nidovirales.

Zoonotic pathogens

   Animal pathogens that can infect and replicate in humans.

Recombination breakpoints

   Distinct sites in the viral genome that are associated with a high frequency of exchange of genetic material between related viruses during co-infection of the same host cell.

Integrins

   Proteins that bind carbohydrate moieties found on proteins of the extracellular matrix or on cell-surface glycoproteins.

Hybridoma

   Clonal cells resulting from the fusion of B lymphoblasts and lymphoid myeloma cells. Hybridoma cells are used for the production of monoclonal antibodies.

Escape mutations

   Nucleotide changes that enable evasion from a selective pressure. Frequently used to describe changes in the viral genome that impair the efficiency of antibodies or antiviral compounds.

Transcription regulatory sequences

   (TRSs). Direct the leader-body junction during synthesis of coronavirus subgenomic RNAs.

Accessory genes

   Sets of coronavirus genes that encode proteins that are neither the non-structural proteins 1–16 (encoded in ORF1a/b and composing the replication and transcription complex) nor the canonical coronavirus structural proteins S, E, M and N. Usually dispensable in cell culture.

Major histocompatibility complex

   Major histocompatibility complex (MHC) molecules are cell-surface proteins that present a repertoire of proteins currently being expressed in the cell or proteins that have been uptaken by the cell in the form of MHC-bound peptides (called epitopes). T cells can recognize the MHC–peptide complexes and are stimulated to exert their immune functions upon recognition of MHC-bound pathogen-derived peptides.

Double-membrane vesicles

   (DMVs). DMVs are characteristic membranous structures induced by coronaviruses and many other positive-sense RNA viruses. They anchor the replication and transcription complex and support viral replication and RNA synthesis.

OAS–RNase L pathway

   There are several cellular 2′,5′-oligoadenylate synthetases that produce 2′,5′-oligoadenylates upon stimulation by double-stranded RNA. 2′,5′-oligoadenylates can stimulate the cellular ribonuclease RNase L that degrades cellular and viral RNAs as part of the antiviral host defence.


https://www.nature.com/articles/s41579-020-00468-6

[Relax]

NB: Aware that posting things 'as is' without personal analysis/commentary is a guideline breach - (and not useful); any discussion of this article will need to cross reference newer findings, analysis since last October.

I'm working towards that (in a word doc) whenever I get a chance.

Initial takeaway: there's a LOT of scientific knowledge needed for a person to have an opinion on the science of epidemiology and pandemics in relation to Coronavirus biology and replication, and the complexity and detail in this article indicates this most clearly.
Not even a basic science degree would be sufficient to make broad public statements about these incredibly thorough investigations without joining this specific research team (Switzerland), or at least speaking with them in person - let alone adopting simplistic 'opinions' garnered from non expert sources.

[Margan]

Australia has fallen into demonic enslavement.
Will your country be next?
https://www.youtube.com/watch?v=H8KScb7XjzE

      one can only hope.

demonic enslavement sounds like efficient catalyst!

You know what..... I have been thinking that maybe we really need to go thru this, Orwellian fascism type of thing in order to make the choice for ascension and 4D out of those dire circumstances... or as someone quoted, we need to hit rock bottom before the reverse can happen and we can go up again.
Because as I see it, no one will save us, there is no external saviour and after all, we did not come here for the picknick and the good times, right?
Insofar you are right, it is efficient catalyst, probably the best there ever was...

Edited to add: it is like - the harsher the circumstances, the more the choice is worth... I find it hard to explain what I mean - you know, like in a computer game? if you have many obstacles to overcome, the satisfaction in the end and the gain / win is greater than if you are winning in a beginners mode. So in that sense, if we are able to choose love in a dystopian fascist environment, it probably gets us rid of a shitload of karma
It is intriguing to me that Germany is also at the forefront in that regard, and we do have fascist karma and lots of Germans seem to thrive in rules and orders.... although Hitler was a useful idiot for UK and USA, the anglo empire was russophobic from the start and it was great for them to have two rivals (Germany and Russia) combat each other

[Nikki]

Australia has fallen into demonic enslavement.
Will your country be next?
https://www.youtube.com/watch?v=H8KScb7XjzE

      one can only hope.

demonic enslavement sounds like efficient catalyst!

You know what..... I have been thinking that maybe we really need to go thru this, Orwellian fascism type of thing in order to make the choice for ascension and 4D out of those dire circumstances... or as someone quoted, we need to hit rock bottom before the reverse can happen and we can go up again.
Because as I see it, no one will save us, there is no external saviour and after all, we did not come here for the picknick and the good times, right?
Insofar you are right, it is efficient catalyst, probably the best there ever was...

Edited to add: it is like - the harsher the circumstances, the more the choice is worth... I find it hard to explain what I mean - you know, like in a computer game? if you have many obstacles to overcome, the satisfaction in the end and the gain / win is greater than if you are winning in a beginners mode. So in that sense, if we are able to choose love in a dystopian fascist environment, it probably gets us rid of a shitload of karma
It is intriguing to me that Germany is also at the forefront in that regard, and we do have fascist karma and lots of Germans seem to thrive in rules and orders.... although Hitler was a useful idiot for UK and USA, the anglo empire was russophobic from the start and it was great for them to have two rivals (Germany and Russia) combat each other

Two documents indicating covid not real. One man won a court case in Alberta Canada and
all mandates are void (second link)

https://greatreject.org/laboratories-can...ive-tests/

https://www.redvoicemedia.com/video/2021...n-alberta/

Thought you may all find this interesting, we know how to create an illusion but so do the elite using our power to manifest.

[Patrick]

STS was really good creating this illusion, succeeding in including millions of regular moms and dads working in healthcare in their conspiration.

I am starting to wonder if I am the only one who knows people working in healthcare...

[tadeus]

This post makes more sense in this thread.

Here is the summary:

There has been an funny lawsuit in germany, that witness that a measles virus is not existing at all.
The evidence is universal to the concept of proofing the existance of an virus.

The lawsuit was about an competition that such an virus does not exist.

OLG Stuttgart Urteil vom 16.2.2016, 12 U 63/15

So this pictures of the COVID-virus are only a model to help us to manifestate it!