veteranstoday--2020-03-23--Pravda: US Army Created COVID 19 in 2015-Research (Proofs or Debunking, You Pick)

The emergence of SARS-CoV heralded a new era in the cross-species transmission of severe respiratory illness with globalization leading to rapid spread around the world and massive economic impact3,4 .
Since then , several strains—including influenza A strains H5N1 , H1N1 and H7N9 and MERS-CoV—have emerged from animal populations , causing considerable disease , mortality and economic hardship for the afflicted regions5 .
Although public health measures were able to stop the SARS-CoV outbreak4 , recent metagenomics studies have identified sequences of closely related SARS-like viruses circulating in Chinese bat populations that may pose a future threat1,6 .
However , @ @ @ @ @ @ @ identify and prepare for future prepandemic viruses .
Therefore , to examine the emergence potential ( that is , the potential to infect humans ) of circulating bat CoVs , we built a chimeric virus encoding a novel , zoonotic CoV spike protein—from the RsSHC014-CoV sequence that was isolated from Chinese horseshoe bats1—in the context of the SARS-CoV mouse-adapted backbone .
The hybrid virus allowed us to evaluate the ability of the novel spike protein to cause disease independently of other necessary adaptive mutations in its natural backbone .
Using this approach , @ @ @ @ @ @ @ SHC014 spike protein in primary human airway cells and in vivo , and tested the efficacy of available immune therapeutics against SHC014-CoV .
Together , the strategy translates metagenomics data to help predict and prepare for future emergent viruses .
The sequences of SHC014 and the related RsWIV1-CoV show that these CoVs are the closest relatives to the epidemic SARS-CoV strains ( Fig .
1a , b ) ; however , there are important differences in the 14 residues that bind human ACE2 , the receptor for SARS-CoV , including the five that @ @ @ @ @ @ @ , L472 , N479 , T487 and Y491 ( ref .
7 ) .
In WIV1 , three of these residues vary from the epidemic SARS-CoV Urbani strain , but they were not expected to alter binding to ACE2 ( Supplementary Fig .
1a , b and Supplementary Table 1 ) .
This fact is confirmed by both pseudotyping experiments that measured the ability of lentiviruses encoding WIV1 spike proteins to enter cells expressing human ACE2 ( Supplementary Fig .
1 ) and by in vitro replication assays of WIV1-CoV ( ref .
@ @ @ @ @ @ @ of 14 ACE2-interaction residues in SHC014 are different from those in SARS-CoV , including all five residues critical for host range ( Supplementary Fig .
1c and Supplementary Table 1 ) .
These changes , coupled with the failure of pseudotyped lentiviruses expressing the SHC014 spike to enter cells ( Supplementary Fig .
1d ) , suggested that the SHC014 spike is unable to bind human ACE2 .
However , similar changes in related SARS-CoV strains had been reported to allow ACE2 binding7,8 , suggesting that additional functional testing was required for verification @ @ @ @ @ @ @ spike in the context of the replication-competent , mouse-adapted SARS-CoV backbone ( we hereafter refer to the chimeric CoV as SHC014-MA15 ) to maximize the opportunity for pathogenesis and vaccine studies in mice ( Supplementary Fig .
2a ) .
Despite predictions from both structure-based modeling and pseudotyping experiments , SHC014-MA15 was viable and replicated to high titers in Vero cells ( Supplementary Fig .
2b ) .
Similarly to SARS , SHC014-MA15 also required a functional ACE2 molecule for entry and could use human , civet and bat ACE2 orthologs ( Supplementary @ @ @ @ @ @ @ To test the ability of the SHC014 spike to mediate infection of the human airway , we examined the sensitivity of the human epithelial airway cell line Calu-3 2B4 ( ref .
9 ) to infection and found robust SHC014-MA15 replication , comparable to that of SARS-CoV Urbani ( Fig .
1c ) .
To extend these findings , primary human airway epithelial ( HAE ) cultures were infected and showed robust replication of both viruses ( Fig .
1d ) .
Together , the data confirm the ability of viruses with the @ @ @ @ @ @ @ and underscore the potential threat of cross-species transmission of SHC014-CoV .
To evaluate the role of the SHC014 spike in mediating infection in vivo , we infected 10-week-old BALB/c mice with 104 plaque-forming units ( p.f.u . )
of either SARS-MA15 or SHC014-MA15 ( Fig .
1e–h ) .
Animals infected with SARS-MA15 experienced rapid weight loss and lethality by 4 d post infection ( d.p.i . )
; in contrast , SHC014-MA15 infection produced substantial weight loss ( 10 % ) but no lethality in mice ( Fig .
1e ) .
@ @ @ @ @ @ @ viral titers from the lungs of mice infected with SARS-MA15 or SHC014-MA15 ( Fig .
1f ) .
Whereas lungs from the SARS-MA15–infected mice showed robust staining in both the terminal bronchioles and the lung parenchyma 2 d.p.i .
( Fig .
1g ) , those of SHC014-MA15–infected mice showed reduced airway antigen staining ( Fig .
1h ) ; in contrast , no deficit in antigen staining was observed in the parenchyma or in the overall histology scoring , suggesting differential infection of lung tissue for SHC014-MA15 ( Supplementary Table 2 ) @ @ @ @ @ @ @ susceptible , aged ( 12-month-old ) animals .
SARS-MA15–infected animals rapidly lost weight and succumbed to infection ( Supplementary Fig .
3a , b ) .
SHC014-MA15 infection induced robust and sustained weight loss , but had minimal lethality .
Trends in the histology and antigen staining patterns that we observed in young mice were conserved in the older animals ( Supplementary Table 3 ) .
We excluded the possibility that SHC014-MA15 was mediating infection through an alternative receptor on the basis of experiments using Ace2−/− mice , which did not show weight @ @ @ @ @ @ @ ( Supplementary Fig .
4a , b and Supplementary Table 2 ) .
Together , the data indicate that viruses with the SHC014 spike are capable of inducing weight loss in mice in the context of a virulent CoV backbone .
Given the preclinical efficacy of Ebola monoclonal antibody therapies , such as ZMApp10 , we next sought to determine the efficacy of SARS-CoV monoclonal antibodies against infection with SHC014-MA15 .
Four broadly neutralizing human monoclonal antibodies targeting SARS-CoV spike protein had been previously reported and are probable reagents for immunotherapy11,12,13 .
We @ @ @ @ @ @ @ viral replication ( expressed as percentage inhibition of viral replication ) and found that whereas wild-type SARS-CoV Urbani was strongly neutralized by all four antibodies at relatively low antibody concentrations ( Fig .
2a–d ) , neutralization varied for SHC014-MA15 .
Fm6 , an antibody generated by phage display and escape mutants11,12 , achieved only background levels of inhibition of SHC014-MA15 replication ( Fig .
2a ) .
Similarly , antibodies 230.15 and 227.14 , which were derived from memory B cells of SARS-CoV–infected patients13 , also failed to block SHC014-MA15 replication ( @ @ @ @ @ @ @ For all three antibodies , differences between the SARS and SHC014 spike amino acid sequences corresponded to direct or adjacent residue changes found in SARS-CoV escape mutants ( fm6 N479R ; 230.15 L443V ; 227.14 K390Q/E ) , which probably explains the absence of the antibodies ’ neutralizing activity against SHC014 .
Finally , monoclonal antibody 109.8 was able to achieve 50 % neutralization of SHC014-MA15 , but only at high concentrations ( 10 μg/ml ) ( Fig .
2d ) .
Together , the results demonstrate that broadly neutralizing antibodies against SARS-CoV @ @ @ @ @ @ @ SARS-like CoV strains such as SHC014 .
To evaluate the efficacy of existing vaccines against infection with SHC014-MA15 , we vaccinated aged mice with double-inactivated whole SARS-CoV ( DIV ) .
Previous work showed that DIV could neutralize and protect young mice from challenge with a homologous virus14 ; however , the vaccine failed to protect aged animals in which augmented immune pathology was also observed , indicating the possibility of the animals being harmed because of the vaccination15 .
Here we found that DIV did not provide protection from challenge with SHC014-MA15 @ @ @ @ @ @ @ titer ( Supplementary Fig .
5a , b ) .
Consistent with a previous report with other heterologous group 2b CoVs15 , serum from DIV-vaccinated , aged mice also failed to neutralize SHC014-MA15 ( Supplementary Fig .
5c ) .
Notably , DIV vaccination resulted in robust immune pathology ( Supplementary Table 4 ) and eosinophilia ( Supplementary Fig .
5d–f ) .
Together , these results confirm that the DIV vaccine would not be protective against infection with SHC014 and could possibly augment disease in the aged vaccinated group .
In contrast @ @ @ @ @ @ @ the use of SHC014-MA15 as a live , attenuated vaccine showed potential cross-protection against challenge with SARS-CoV , but the results have important caveats .
We infected young mice with 104 p.f.u .
of SHC014-MA15 and observed them for 28 d. We then challenged the mice with SARS-MA15 at day 29 ( Supplementary Fig .
6a ) .
The prior infection of the mice with the high dose of SHC014-MA15 conferred protection against challenge with a lethal dose of SARS-MA15 , although there was only a minimal SARS-CoV neutralization response from the antisera @ @ @ @ @ @ @ Supplementary Fig .
6b , 1:200 ) .
In the absence of a secondary antigen boost , 28 d.p.i .
represents the expected peak of antibody titers and implies that there will be diminished protection against SARS-CoV over time16,17 .
Similar results showing protection against challenge with a lethal dose of SARS-CoV were observed in aged BALB/c mice with respect to weight loss and viral replication ( Supplementary Fig .
6c , d ) .
However , the SHC014-MA15 infection dose of 104 p.f.u .
induced > 10 % weight loss and lethality @ @ @ @ @ @ @ 1 and Supplementary Fig .
3 ) .
We found that vaccination with a lower dose of SHC014-MA15 ( 100 p.f.u . )
, did not induce weight loss , but it also failed to protect aged animals from a SARS-MA15 lethal dose challenge ( Supplementary Fig .
6e , f ) .
Together , the data suggest that SHC014-MA15 challenge may confer cross-protection against SARS-CoV through conserved epitopes , but the required dose induces pathogenesis and precludes use as an attenuated vaccine .
Having established that the SHC014 spike has the ability @ @ @ @ @ @ @ cause disease in mice , we next synthesized a full-length SHC014-CoV infectious clone based on the approach used for SARS-CoV ( Fig .
3a ) 2 .
Replication in Vero cells revealed no deficit for SHC014-CoV relative to that for SARS-CoV ( Fig .
3b ) ; however , SHC014-CoV was significantly ( P < 0.01 ) attenuated in primary HAE cultures at both 24 and 48 h after infection ( Fig .
3c ) .
In vivo infection of mice demonstrated no significant weight loss but showed reduced viral replication in lungs @ @ @ @ @ @ @ to SARS-CoV Urbani ( Fig .
3d , e ) .
Together , the results establish the viability of full-length SHC014-CoV , but suggest that further adaptation is required for its replication to be equivalent to that of epidemic SARS-CoV in human respiratory cells and in mice .
During the SARS-CoV epidemic , links were quickly established between palm civets and the CoV strains that were detected in humans4 .
Building on this finding , the common emergence paradigm argues that epidemic SARS-CoV originated as a bat virus , jumped to civets and @ @ @ @ @ @ @ RBD ) to improve binding to civet Ace2 ( ref .
18 ) .
Subsequent exposure to people in live-animal markets permitted human infection with the civet strain , which , in turn , adapted to become the epidemic strain ( Fig .
4a ) .
However , phylogenetic analysis suggests that early human SARS strains appear more closely related to bat strains than to civet strains18 .
Therefore , a second paradigm argues that direct bat-human transmission initiated SARS-CoV emergence and that palm civets served as a secondary host and reservoir for @ @ @ @ @ @ @ 19 .
For both paradigms , spike adaptation in a secondary host is seen as a necessity , with most mutations expected to occur within the RBD , thereby facilitating improved infection .
Both theories imply that pools of bat CoVs are limited and that host-range mutations are both random and rare , reducing the likelihood of future emergence events in humans .
Although our study does not invalidate the other emergence routes , it does argue for a third paradigm in which circulating bat CoV pools maintain ‘ poised ’ spike proteins @ @ @ @ @ @ @ mutation or adaptation ( Fig .
4c ) .
This hypothesis is illustrated by the ability of a chimeric virus containing the SHC014 spike in a SARS-CoV backbone to cause robust infection in both human airway cultures and in mice without RBD adaptation .
Coupled with the observation of previously identified pathogenic CoV backbones3,20 , our results suggest that the starting materials required for SARS-like emergent strains are currently circulating in animal reservoirs .
Notably , although full-length SHC014-CoV probably requires additional backbone adaption to mediate human disease , the documented high-frequency recombination @ @ @ @ @ @ @ of future emergence and the need for further preparation .
To date , genomics screens of animal populations have primarily been used to identify novel viruses in outbreak settings21 .
The approach here extends these data sets to examine questions of viral emergence and therapeutic efficacy .
We consider viruses with the SHC014 spike a potential threat owing to their ability to replicate in primary human airway cultures , the best available model for human disease .
In addition , the observed pathogenesis in mice indicates a capacity for SHC014-containing viruses to cause @ @ @ @ @ @ @ adaptation .
Notably , differential tropism in the lung as compared to that with SARS-MA15 and attenuation of full-length SHC014-CoV in HAE cultures relative to SARS-CoV Urbani suggest that factors beyond ACE2 binding—including spike processivity , receptor bio-availability or antagonism of the host immune responses—may contribute to emergence .
However , further testing in nonhuman primates is required to translate these finding into pathogenic potential in humans .
Importantly , the failure of available therapeutics defines a critical need for further study and for the development of treatments .
With this knowledge , @ @ @ @ @ @ @ treatments can be produced that are protective against the emergence of group 2b–specific CoVs , such as SHC014 , and these can be applied to other CoV branches that maintain similarly heterogeneous pools .
In addition to offering preparation against future emerging viruses , this approach must be considered in the context of the US government–mandated pause on gain-of-function ( GOF ) studies22 .
On the basis of previous models of emergence ( Fig .
4a , b ) , the creation of chimeric viruses such as SHC014-MA15 was not expected to increase @ @ @ @ @ @ @ to its parental mouse-adapted SARS-CoV , similar studies examining the pathogenicity of CoVs with the wild-type Urbani spike within the MA15 backbone showed no weight loss in mice and reduced viral replication23 .
Thus , relative to the Urbani spike–MA15 CoV , SHC014-MA15 shows a gain in pathogenesis ( Fig .
1 ) .
On the basis of these findings , scientific review panels may deem similar studies building chimeric viruses based on circulating strains too risky to pursue , as increased pathogenicity in mammalian models can not be excluded .
Coupled with @ @ @ @ @ @ @ of monoclonal antibodies using escape mutants , research into CoV emergence and therapeutic efficacy may be severely limited moving forward .
Together , these data and restrictions represent a crossroads of GOF research concerns ; the potential to prepare for and mitigate future outbreaks must be weighed against the risk of creating more dangerous pathogens .
In developing policies moving forward , it is important to consider the value of the data generated by these studies and whether these types of chimeric virus studies warrant further investigation versus the inherent risks involved .
@ @ @ @ @ @ @ data to identify a potential threat posed by the circulating bat SARS-like CoV SHC014 .
Because of the ability of chimeric SHC014 viruses to replicate in human airway cultures , cause pathogenesis in vivo and escape current therapeutics , there is a need for both surveillance and improved therapeutics against circulating SARS-like viruses .
Our approach also unlocks the use of metagenomics data to predict viral emergence and to apply this knowledge in preparing to treat future emerging virus infections .

Normally, Rand Paul is a clown with his war on Fauci, always for the wrong reasons mind you. Generally, Rand Paul despite his medical background, sounds like a moron when talking science.
Rand Paul runs to Wuhan instead of a dozen American-funded labs, some here, some in Georgia, Romania, even Libya, that have Level 4 capabilities and USAID/CIA funding as we prove below.
Paul is now running the coverup.
Today he is reading something he didn’t write, something given him by his masters, and those masters, it now seems beyond any doubt, are using COVID 19 to destroy the US.
We believe Rand Paul is very much a part of it.
Watch Rand Paul lie, over and over and over, but carefully, reading a skillfully crafted disinformation packet that is filled with half-truths and utter lies.
The public is supposed to be too stupid to understand.
VT had the story right from the beginning. Now that the CIA is officially tasked with finding a source for COVID, someone to blame, the vast American program of bio-warfare defense which creates every nasty virus we have seen, probably ebola, certainly swine flu, and then “accidentally” release them in such a way that the CIA’s agenda is served.
Since this article was written, we have begun to look at the worldwide operations of US nuclear/bio/chem contractor, Kushner-Trump’s favorite, Battelle, and their secret labs around the world.
When we began, our people started to be threatened. That was a serious mistake.
Submit this paper to any physician or other qualified bio-sciences specialist. See what they say.
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Introduction
Documents below will show that research to create COVID 19 began in the United States in 2006 and culminated in a successful bio-weapon in 2015, with work done at the University of North Carolina and at Harvard, and at the Food and Drug Administration’s lab in Arkansas.
Their work was titled:
A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence
They did this and more, so much more as you will read below.
As Trump said, over and over and over, the Chinese were involved.
Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China supplied the Wuhan Bat Virus which was used in the American study. Their name was included for that reason only.
COVID 19 was a US Army bio-weapons project to manufacture a pneumonia-causing disease that would be nearly impossible to vaccinate for in patients over 40 years old.
The proof is here, simply scroll down. The study was run by the University of North Carolina and funded by USAID/CIA. It chose a Chinese bat virus and chose to include a medical facility in Wuhan as well.
Now we know why, a smokescreen of the blame for a program China had little or nothing to do with, something satanically evil and purely American.
In November 2015, a study was published outlining the capability of producing the virus we are dealing with now. Among the many involved was a lab in Wuhan, China. It was listed from the beginning as one of the dozens, mostly American, working on this project.
However, one key participant was left out, USAID. It is suspected, deeply so, that USAID is a front for American bio-warfare research such as that done in Tbilisi, Georgia, and elsewhere, much documented. This is the citation that adds USAID to the research funding group.
Change history
20 November 2015
In the version of this article initially published online, the authors omitted to acknowledge a funding source, USAID-EPT-PREDICT funding from EcoHealth Alliance, to Z.-L.S. The error has been corrected for the print, PDF and HTML versions of this article.
[ Editor’s Note: We will now present Pravda’s biased article and, below that, the actual study proving the capability of producing COVID 19, proving it is not a naturally occurring virus once and for all.
As to who did what, this is not our job but we are proving, categorically, that when a Chinese lab is mentioned, it is a minor player in an American effort, as outlined exhaustively below.
This makes discussing the Wuhan lab possibly complicit in bio-warfare.
Similarly, when Forbes Magazine and others stated they could prove COVID 19 was made naturally, and of course, they had the same access we have, we suspect that they are part of a disinformation effort tied to USAID and bio-warfare.
Suspicion is not proof. The proof is proof and there is proof enough to drown in. Our thanks to the American medical professionals who pimped themselves out to the US Army and CIA and who helped bring us where we are now, a nation broken to pieces…VeteransToday ]
Pravda.Ru: Such material appeared in 2015 on the website of the scientific journal Natura 2015. Then the authors claimed that after the advent of the SARS virus (2002-2003) and the Middle East respiratory syndrome (MERS), scientists were aware of the risk of interspecific transmission that would lead to an epidemic among people.
Successful lab experiment
Among other things, the research team studied bats, which are the largest incubators of coronaviruses. Nevertheless, bats could not transmit the coronavirus to humans because they could not interact with human cells with ACE2 receptors.
The material also stated that horseshoe bats carry a strain of SARS coronavirus that can be transmitted to humans. It has been named the SHC014-CoV virus.
To better study this virus, scientists copied the coronavirus and infected it with laboratory mice. The results showed that the virus is really able to bind to human cells with ACE2 receptors and multiply in the cells of the respiratory system.
In the research work, it is noted that laboratory materials, samples and equipment that were used in the research were obtained from the Army Medical Research Institute of Infectious Diseases. Although it is not yet possible to say for sure that the virus that was tested in laboratory mice is the same as the SARS-Cove-2 coronavirus.
NATO policy
However, interesting things can be found in earlier documents.
For example:
The 2019 Alliance’s activity report says that in 2019, the Alliance’s first place in research and development was occupied by the topic of radiochemical and biological protection (29%), shifting the seemingly most pressing problem of Europe – counterterrorism (it turned out to be 4- m priority).
A year earlier, in 2018, the situation was exactly the opposite: terrorism, as it should be, was in the first place (28%), and radiochemical and biological protection in the fourth (13%).
As the Brussels snitch writes in the telegram channel, “given the absence of visible reasons for such a sharp change in scientific interests, there are two options and both are unpleasant:
or NATO now wags the fifth point, falsifying the data to show “and we always prepared for viruses, we are modern”,
or even in 2019 in the alliance, God forgive me, they knew where the trouble would come from.
Yes, the first option is much more real, but, you see, the facts are surprising.
Source: Pravda
Original 2015 Research Unedited and Complete
Published: 09 November 2015 A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence by Vineet D Menachery, Boyd L Yount Jr, Kari Debbink, Sudhakar Agnihothram, Lisa E Gralinski, Jessica A Plante, Rachel L Graham, Trevor Scobey, Xing-Yi Ge, Eric F Donaldson, Scott H Randell, Antonio Lanzavecchia, Wayne A Marasco, Zhengli-Li Shi, Ralph S Baric
Nature Medicine volume 21, pages 1508–1513 (2015)
A Corrigendum to this article was published on 06 April 2016
This article has been updated
Abstract
The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations1. Using the SARS-CoV reverse genetics system2, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone.
The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin-converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis.
Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approach failed to neutralize and protect from infection with CoVs using the novel spike protein.
On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.
Main
The emergence of SARS-CoV heralded a new era in the cross-species transmission of severe respiratory illness with globalization leading to rapid spread around the world and massive economic impact3,4. Since then, several strains—including influenza A strains H5N1, H1N1 and H7N9, and MERS-CoV—have emerged from animal populations, causing considerable disease, mortality and economic hardship for the afflicted regions5. Although public health measures were able to stop the SARS-CoV outbreak4, recent metagenomics studies have identified sequences of closely related SARS-like viruses circulating in Chinese bat populations that may pose a future threat1,6.
However, sequence data alone provides minimal insights to identify and prepare for future pre-pandemic viruses. Therefore, to examine the emergence potential (that is, the potential to infect humans) of circulating bat CoVs, we built a chimeric virus encoding a novel, zoonotic CoV spike protein—from the RsSHC014-CoV sequence that was isolated from Chinese horseshoe bats1—in the context of the SARS-CoV mouse-adapted backbone. The hybrid virus allowed us to evaluate the ability of the novel spike protein to cause disease independently of other necessary adaptive mutations in its natural backbone.
Using this approach, we characterized CoV infection mediated by the SHC014 spike protein in primary human airway cells and in vivo and tested the efficacy of available immune therapeutics against SHC014-CoV. Together, the strategy translates metagenomics data to help predict and prepare for future emergent viruses.
The sequences of SHC014 and the related RsWIV1-CoV show that these CoVs are the closest relatives to the epidemic SARS-CoV strains (Fig. 1a,b); however, there are important differences in the 14 residues that bind human ACE2, the receptor for SARS-CoV, including the five that are critical for host range: Y442, L472, N479, T487 and Y491 (ref. 7).
In WIV1, three of these residues vary from the epidemic SARS-CoV Urbani strain, but they were not expected to alter binding to ACE2 (Supplementary Fig. 1a,b and Supplementary Table 1). This fact is confirmed by both pseudotyping experiments that measured the ability of lentiviruses encoding WIV1 spike proteins to enter cells expressing human ACE2 (Supplementary Fig. 1) and by in vitro replication assays of WIV1-CoV (ref. 1). In contrast, 7 of 14 ACE2-interaction residues in SHC014 are different from those in SARS-CoV, including all five residues critical for the host range (Supplementary Fig. 1c and Supplementary Table 1).
These changes, coupled with the failure of pseudotyped lentiviruses expressing the SHC014 spike to enter cells (Supplementary Fig. 1d), suggested that the SHC014 spike is unable to bind human ACE2. However, similar changes in related SARS-CoV strains had been reported to allow ACE2 binding7,8, suggesting that additional functional testing was required for verification.
Therefore, we synthesized the SHC014 spike in the context of the replication-competent, mouse-adapted SARS-CoV backbone (we hereafter refer to the chimeric CoV as SHC014-MA15) to maximize the opportunity for pathogenesis and vaccine studies in mice (Supplementary Fig. 2a). Despite predictions from both structure-based modeling and pseudotyping experiments, SHC014-MA15 was viable and replicated to high titers in Vero cells (Supplementary Fig. 2b). Similar to SARS, SHC014-MA15 also required a functional ACE2 molecule for entry and could use human, civet and bat ACE2 orthologs (Supplementary Fig. 2c,d).
To test the ability of the SHC014 spike to mediate infection of the human airway, we examined the sensitivity of the human epithelial airway cell line Calu-3 2B4 (ref. 9) to infection and found robust SHC014-MA15 replication, comparable to that of SARS-CoV Urbani (Fig. 1c).
To extend these findings, primary human airway epithelial (HAE) cultures were infected and showed robust replication of both viruses (Fig. 1d). Together, the data confirm the ability of viruses with the SHC014 spike to infect human airway cells and underscore the potential threat of cross-species transmission of SHC014-CoV.
Figure 1: SARS-like viruses replicate in human airway cells and produce in vivo pathogenesis.
(a) The full-length genome sequences of representative CoVs were aligned and phylogenetically mapped as described in the Online Methods. The scale bar represents nucleotide substitutions, with only bootstrap support above 70% being labeled. The tree shows CoVs divided into three distinct phylogenetic groups, defined as α-CoVs, β-CoVs and γ-CoVs. Classical subgroup clusters are marked as 2a, 2b, 2c and 2d for the β-CoVs and as 1a and 1b for the α-CoVs. (b) Amino acid sequences of the S1 domains of the spikes of representative β-CoVs of the 2b group, including SARS-CoV, were aligned and phylogenetically mapped. The scale bar represents the amino acid substitutions. (c,d) Viral replication of SARS-CoV Urbani (black) and SHC014-MA15 (green) after infection of Calu-3 2B4 cells (c) or well-differentiated, primary air-liquid interface HAE cell cultures (d) at a multiplicity of infection (MOI) of 0.01 for both cell types. Samples were collected at individual time points with biological replicates (n = 3) for both Calu-3 and HAE experiments. (e,f) Weight loss (n = 9 for SARS-CoV MA15; n = 16 for SHC014-MA15) (e) and viral replication in the lungs (n = 3 for SARS-CoV MA15; n = 4 for SHC014-MA15) (f) of 10-week-old BALB/c mice infected with 1 × 104 p.f.u. of mouse-adapted SARS-CoV MA15 (black) or SHC014-MA15 (green) via the intranasal (i.n.) route. (g,h) Representative images of lung sections stained for SARS-CoV N antigen from mice infected with SARS-CoV MA15 (n = 3 mice) (g) or SHC014-MA15 (n = 4 mice) (h) are shown. For each graph, the center value represents the group mean, and the error bars define the s.e.m. Scale bars, 1 mm.
Full size image
To evaluate the role of the SHC014 spike in mediating infection in vivo, we infected 10-week-old BALB/c mice with 104 plaque-forming units (p.f.u.) of either SARS-MA15 or SHC014-MA15 (Fig. 1e–h). Animals infected with SARS-MA15 experienced rapid weight loss and lethality by 4 d post-infection (d.p.i.); in contrast, SHC014-MA15 infection produced substantial weight loss (10%) but no lethality in mice (Fig. 1e). Examination of viral replication revealed nearly equivalent viral titers from the lungs of mice infected with SARS-MA15 or SHC014-MA15 (Fig. 1f). Whereas lungs from the SARS-MA15–infected mice showed robust staining in both the terminal bronchioles and the lung parenchyma 2 d.p.i. (Fig. 1g), those of SHC014-MA15–infected mice showed reduced airway antigen staining (Fig. 1h); in contrast, no deficit in antigen staining was observed in the parenchyma or in the overall histology scoring, suggesting differential infection of lung tissue for SHC014-MA15 (Supplementary Table 2).
We next analyzed infection in more susceptible, aged (12-month-old) animals. SARS-MA15–infected animals rapidly lost weight and succumbed to infection (Supplementary Fig. 3a,b). SHC014-MA15 infection-induced robust and sustained weight loss, but had minimal lethality. Trends in the histology and antigen staining patterns that we observed in young mice were conserved in the older animals (Supplementary Table 3). We excluded the possibility that SHC014-MA15 was mediating infection through an alternative receptor on the basis of experiments using Ace2−/− mice, which did not show weight loss or antigen staining after SHC014-MA15 infection (Supplementary Fig. 4a,b and Supplementary Table 2). Together, the data indicate that viruses with the SHC014 spike are capable of inducing weight loss in mice in the context of a virulent CoV backbone.
Given the preclinical efficacy of Ebola monoclonal antibody therapies, such as ZMApp10, we next sought to determine the efficacy of SARS-CoV monoclonal antibodies against infection with SHC014-MA15. Four broadly neutralizing human monoclonal antibodies targeting SARS-CoV spike protein had been previously reported and are probable reagents for immunotherapy11,12,13. We examined the effect of these antibodies on viral replication (expressed as percentage inhibition of viral replication) and found that whereas wild-type SARS-CoV Urbani was strongly neutralized by all four antibodies at relatively low antibody concentrations (Fig. 2a–d), neutralization varied for SHC014-MA15. Fm6, an antibody generated by phage display and escape mutants11,12, achieved only background levels of inhibition of SHC014-MA15 replication (Fig. 2a). Similarly, antibodies 230.15 and 227.14, which were derived from memory B cells of SARS-CoV–infected patients13, also failed to block SHC014-MA15 replication (Fig. 2b,c). For all three antibodies, differences between the SARS and SHC014 spike amino acid sequences corresponded to direct or adjacent residue changes found in SARS-CoV escape mutants (fm6 N479R; 230.15 L443V; 227.14 K390Q/E), which probably explains the absence of the antibodies’ neutralizing activity against SHC014. Finally, monoclonal antibody 109.8 was able to achieve 50% neutralization of SHC014-MA15, but only at high concentrations (10 μg/ml) (Fig. 2d). Together, the results demonstrate that broadly neutralizing antibodies against SARS-CoV may only have marginal efficacy against emergent SARS-like CoV strains such as SHC014.
Figure 2: SARS-CoV monoclonal antibodies have marginal efficacy against SARS-like CoVs.
(a–d) Neutralization assays evaluating efficacy (measured as a reduction in the number of plaques) of a panel of monoclonal antibodies, which were all originally generated against epidemic SARS-CoV, against infection of Vero cells with SARS-CoV Urbani (black) or SHC014-MA15 (green). The antibodies tested were fm6 (n = 3 for Urbani; n = 5 for SHC014-MA15)11,12 (a), 230.15 (n = 3 for Urbani; n = 2 for SHC014-MA15) (b), 227.15 (n = 3 for Urbani; n = 5 for SHC014-MA15) (c) and 109.8 (n = 3 for Urbani; n = 2 for SHC014-MA15)13 (d). Each data point represents the group mean and error bars define the s.e.m. Note that the error bars in SARS-CoV Urbani–infected Vero cells in b,c are overlapped by the symbols and are not visible.
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To evaluate the efficacy of existing vaccines against infection with SHC014-MA15, we vaccinated aged mice with double-inactivated whole SARS-CoV (DIV). Previous work showed that DIV could neutralize and protect young mice from challenge with a homologous virus14; however, the vaccine failed to protect aged animals in which augmented immune pathology was also observed, indicating the possibility of the animals being harmed because of the vaccination15. Here we found that DIV did not provide protection from challenge with SHC014-MA15 with regards to weight loss or viral titer (Supplementary Fig. 5a,b). Consistent with a previous report with other heterologous groups 2b CoVs15, serum from DIV-vaccinated, aged mice also failed to neutralize SHC014-MA15 (Supplementary Fig. 5c).
Notably, DIV vaccination resulted in robust immune pathology (Supplementary Table 4) and eosinophilia (Supplementary Fig. 5d–f). Together, these results confirm that the DIV vaccine would not be protective against infection with SHC014 and could possibly augment disease in the aged vaccinated group.
In contrast to the vaccination of mice with DIV, the use of SHC014-MA15 as a live, attenuated vaccine showed potential cross-protection against challenge with SARS-CoV, but the results have important caveats. We infected young mice with 104 p.f.u. of SHC014-MA15 and observed them for 28 d. We then challenged the mice with SARS-MA15 at day 29 (Supplementary Fig. 6a). The prior infection of the mice with the high dose of SHC014-MA15 conferred protection against challenge with a lethal dose of SARS-MA15, although there was only a minimal SARS-CoV neutralization response from the antisera elicited 28 d after SHC014-MA15 infection (Supplementary Fig. 6b, 1:200). In the absence of a secondary antigen boost, 28 d.p.i. represents the expected peak of antibody titers and implies that there will be diminished protection against SARS-CoV over time16,17. Similar results showing protection against challenge with a lethal dose of SARS-CoV were observed in aged BALB/c mice with respect to weight loss and viral replication (Supplementary Fig. 6c,d). However, the SHC014-MA15 infection dose of 104 p.f.u. induced >10% weight loss and lethality in some aged animals (Fig. 1 and Supplementary Fig. 3). We found that vaccination with a lower dose of SHC014-MA15 (100 p.f.u.), did not induce weight loss, but it also failed to protect aged animals from a SARS-MA15 lethal dose challenge (Supplementary Fig. 6e,f). Together, the data suggest that the SHC014-MA15 challenge may confer cross-protection against SARS-CoV through conserved epitopes, but the required dose induces pathogenesis and precludes use as an attenuated vaccine.
Having established that the SHC014 spike has the ability to mediate infection of human cells and cause disease in mice, we next synthesized a full-length SHC014-CoV infectious clone based on the approach used for SARS-CoV (Fig. 3a)2. Replication in Vero cells revealed no deficit for SHC014-CoV relative to that for SARS-CoV (Fig. 3b); however, SHC014-CoV was significantly (P < 0.01) attenuated in primary HAE cultures at both 24 and 48 h after infection (Fig. 3c). In vivo infection of mice demonstrated no significant weight loss but showed reduced viral replication in lungs of full-length SHC014-CoV infection, as compared to SARS-CoV Urbani (Fig. 3d,e). Together, the results establish the viability of full-length SHC014-CoV but suggest that further adaptation is required for its replication to be equivalent to that of epidemic SARS-CoV in human respiratory cells and in mice.
Figure 3: Full-length SHC014-CoV replicates in human airways but lacks the virulence of epidemic SARS-CoV.
(a) Schematic of the SHC014-CoV molecular clone, which was synthesized as six contiguous cDNAs (designated SHC014A, SHC014B, SHC014C, SHC014D, SHC014E and SHC014F) flanked by unique BglI sites that allowed for directed assembly of the full-length cDNA expressing open reading frames (for 1a, 1b, spike, 3, envelope, matrix, 6–8 and nucleocapsid). Underlined nucleotides represent the overhang sequences formed after restriction enzyme cleavage. (b,c) Viral replication of SARS-CoV Urbani (black) or SHC014-CoV (green) after infection of Vero cells (b) or well-differentiated, primary air-liquid interface HAE cell cultures (c) at an MOI of 0.01. Samples were collected at individual time points with biological replicates (n = 3) for each group. Data represent one experiment for both Vero and HAE cells. (d,e) Weight loss (n = 3 for SARS-CoV MA15, n = 7 for SHC014-CoV; n = 6 for SARS-Urbani) (d) and viral replication in the lungs (n = 3 for SARS-Urbani and SHC014-CoV) (e) of 10-week-old BALB/c mice infected with 1 × 105 p.f.u. of SARS-CoV MA15 (gray), SHC014-CoV (green) or SARS-CoV Urbani (black) via the i.n. route. Each data point represents the group mean, and error bars define the s.e.m. **P < 0.01 and ***P < 0.001 using two-tailed Student’s t-test of individual time points.
During the SARS-CoV epidemic, links were quickly established between palm civets and the CoV strains that were detected in humans4.
Building on this finding, the common emergence paradigm argues that epidemic SARS-CoV originated as a bat virus, jumped to civets, and incorporated changes within the receptor-binding domain (RBD) to improve binding to civet Ace2 (ref. 18).
Subsequent exposure to people in live-animal markets permitted human infection with the civet strain, which, in turn, adapted to become the epidemic strain (Fig. 4a).
However, phylogenetic analysis suggests that early human SARS strains appear more closely related to bat strains than to civet strains18.
Therefore, a second paradigm argues that direct bat-human transmission initiated SARS-CoV emergence and that palm civets served as a secondary host and reservoir for continued infection (Fig. 4b)19. For both paradigms, spike adaptation in a secondary host is seen as a necessity, with most mutations expected to occur within the RBD, thereby facilitating improved infection. Both theories imply that pools of bat CoVs are limited and that host-range mutations are both random and rare, reducing the likelihood of future emergence events in humans.
Figure 4: Emergence paradigms for coronaviruses.
Coronavirus strains are maintained in quasi-species pools circulating in bat populations. (a,b) Traditional SARS-CoV emergence theories posit that host-range mutants (red circle) represent random and rare occurrences that permit infection of alternative hosts. The secondary-host paradigm (a) argues that a nonhuman host is infected by a bat progenitor virus and, through adaptation, facilitates transmission to humans; subsequent replication in humans leads to the epidemic viral strain. The direct paradigm (b) suggests that transmission occurs between bats and humans without the requirement of an intermediate host; selection then occurs in the human population with closely related viruses replicating in a secondary host, permitting continued viral persistence and adaptation in both. (c) The data from chimeric SARS-like viruses argue that the quasi-species pools maintain multiple viruses capable of infecting human cells without the need for mutations (red circles). Although adaptations in secondary or human hosts may be required for epidemic emergence, if SHC014 spike–containing viruses recombined with virulent CoV backbones (circles with green outlines), then epidemic disease may be the result in humans. Existing data support elements of all three paradigms.
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Although our study does not invalidate the other emergence routes, it does argue for a third paradigm in which circulating bat CoV pools maintain ‘poised’ spike proteins that are capable of infecting humans without mutation or adaptation (Fig. 4c). This hypothesis is illustrated by the ability of a chimeric virus containing the SHC014 spike in a SARS-CoV backbone to cause robust infection in both human airway cultures and in mice without RBD adaptation.
Coupled with the observation of previously identified pathogenic CoV backbones3,20, our results suggest that the starting materials required for SARS-like emergent strains are currently circulating in animal reservoirs. Notably, although full-length SHC014-CoV probably requires additional backbone adaption to mediate human disease, the documented high-frequency recombination events in CoV families underscores the possibility of future emergence and the need for further preparation.
To date, genomics screens of animal populations have primarily been used to identify novel viruses in outbreak settings21. The approach here extends these data sets to examine questions of viral emergence and therapeutic efficacy. We consider viruses with the SHC014 spike a potential threat owing to their ability to replicate in primary human airway cultures, the best available model for human disease. In addition, the observed pathogenesis in mice indicates a capacity for SHC014-containing viruses to cause disease in mammalian models, without RBD adaptation.
Notably, differential tropism in the lung as compared to that with SARS-MA15 and attenuation of full-length SHC014-CoV in HAE cultures relative to SARS-CoV Urbani suggest that factors beyond ACE2 binding—including spike processivity, receptor bio-availability or antagonism of the host immune responses—may contribute to the emergence. However, further testing in nonhuman primates is required to translate these findings into the pathogenic potential in humans.
Importantly, the failure of available therapeutics defines a critical need for further study and for the development of treatments. With this knowledge, surveillance programs, diagnostic reagents, and effective treatments can be produced that are protective against the emergence of group 2b–specific CoVs, such as SHC014, and these can be applied to other CoV branches that maintain similarly heterogeneous pools.
In addition to offering preparation against future emerging viruses, this approach must be considered in the context of the US government-mandated pause on gain-of-function (GOF) studies22.
On the basis of previous models of emergence (Fig. 4a,b), the creation of chimeric viruses such as SHC014-MA15 was not expected to increase pathogenicity. Although SHC014-MA15 is attenuated relative to its parental mouse-adapted SARS-CoV, similar studies examining the pathogenicity of CoVs with the wild-type Urbani spike within the MA15 backbone showed no weight loss in mice and reduced viral replication23. Thus, relative to the Urbani spike–MA15 CoV, SHC014-MA15 shows again in pathogenesis (Fig. 1).
On the basis of these findings, scientific review panels may deem similar studies building chimeric viruses based on circulating strains too risky to pursue, as increased pathogenicity in mammalian models cannot be excluded.
Coupled with restrictions on mouse-adapted strains and the development of monoclonal antibodies using escape mutants, research into CoV emergence and therapeutic efficacy may be severely limited moving forward. Together, these data and restrictions represent a crossroads of GOF research concerns; the potential to prepare for and mitigate future outbreaks must be weighed against the risk of creating more dangerous pathogens. In developing policies moving forward, it is important to consider the value of the data generated by these studies and whether these types of chimeric virus studies warrant further investigation versus the inherent risks involved.
Overall, our approach has used metagenomics data to identify a potential threat posed by the circulating bat SARS-like CoV SHC014. Because of the ability of chimeric SHC014 viruses to replicate in human airway cultures, cause pathogenesis in vivo and escape current therapeutics, there is a need for both surveillance and improved therapeutics against circulating SARS-like viruses. Our approach also unlocks the use of metagenomics data to predict viral emergence and to apply this knowledge in preparing to treat future emerging virus infections.
Methods
Viruses, cells, in vitro infection and plaque assays
Wild-type SARS-CoV (Urbani), mouse-adapted SARS-CoV (MA15) and chimeric SARS-like CoVs were cultured on Vero E6 cells (obtained from United States Army Medical Research Institute of Infectious Diseases), grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, CA) and 5% fetal clone serum (FCS) (Hyclone, South Logan, UT) along with antibiotic/antimycotic (Gibco, Carlsbad, CA). DBT cells (Baric laboratory, source unknown) expressing ACE2 orthologs have been previously described for both human and civet; bat Ace2 sequence was based on that from Rhinolophus leschenaulti, and DBT cells expressing bat Ace2 were established as described previously8.
Pseudotyping experiments were similar to those using an HIV-based pseudovirus, prepared as previously described10, and examined on HeLa cells (Wuhan Institute of Virology) that expressed ACE2 orthologs. HeLa cells were grown in minimal essential medium (MEM) (Gibco, CA) supplemented with 10% FCS (Gibco, CA) as previously described24.
Growth curves in Vero E6, DBT, Calu-3 2B4, and primary human airway epithelial cells were performed as previously described8,25. None of the working cell line stocks were authenticated or tested for mycoplasma recently, although the original seed stocks used to create the working stocks are free from contamination. Human lungs for HAE cultures were procured under the University of North Carolina at Chapel Hill Institutional Review Board–approved protocols. HAE cultures represent highly differentiated human airway epithelium containing ciliated and non-ciliated epithelial cells as well as goblet cells. The cultures are also grown on an air-liquid interface for several weeks before use, as previously described26.
Briefly, cells were washed with PBS and inoculated with the virus or mock-diluted in PBS for 40 min at 37 °C. After inoculation, cells were washed three times and a fresh medium was added to signify time ‘0’. Three or more biological replicates were harvested at each described time point. No blinding was used in any sample collections nor was samples randomized. All virus cultivation was performed in a biosafety level (BSL) 3 laboratory with redundant fans in the biosafety cabinets, as described previously by our group2. All personnel wore powered air-purifying respirators (Breathe Easy, 3M) with Tyvek suits, aprons, and booties and were double-gloved.
Sequence clustering and structural modeling.
The full-length genomic sequences and the amino acid sequences of the S1 domains of the spike of representative CoVs were downloaded from Genbank or Pathosystems Resource Integration Center (PATRIC), aligned with ClustalX and phylogenetically compared by using maximum likelihood estimation using 100 bootstraps or by using the PhyML package, respectively. The tree was generated using maximum likelihood with the PhyML package. The scale bar represents nucleotide substitutions. Only nodes with bootstrap support above 70% are labeled.
The tree shows that CoVs are divided into three distinct phylogenetic groups defined as α-CoVs, β-CoVs, and γ-CoVs. Classical subgroup clusters are marked as 2a, 2b, 2c, and 2d for β-CoVs, and 1a and 1b for the α-CoVs. Structural models were generated using Modeller (Max Planck Institute Bioinformatics Toolkit) to generate homology models for SHC014 and Rs3367 of the SARS RBD in complex with ACE2 based on crystal structure 2AJF (Protein Data Bank). Homology models were visualized and manipulated in MacPyMol (version 1.3).
Construction of SARS-like chimeric viruses.
Both wild-type and chimeric viruses were derived from either SARS-CoV Urbani or the corresponding mouse-adapted (SARS-CoV MA15) infectious clone (ic) as previously described27.
Plasmids containing spike sequences for SHC014 were extracted by restriction digest and ligated into the E and F plasmid of the MA15 infectious clone. The clone was designed and purchased from Bio Basic as six contiguous cDNAs using published sequences flanked by unique class II restriction endonuclease sites (BglI). Thereafter, plasmids containing wild-type, chimeric SARS-CoV, and SHC014-CoV genome fragments were amplified, excised, ligated, and purified.
In vitro transcription reactions were then performed to synthesize full-length genomic RNA, which was transfected into Vero E6 cells as previously described2. The medium from transfected cells was harvested and served as seed stocks for subsequent experiments. Chimeric and full-length viruses were confirmed by sequence analysis before use in these studies. Synthetic construction of chimeric mutant and full-length SHC014-CoV was approved by the University of North Carolina Institutional Biosafety Committee and the Dual Use Research of Concern committee.
Ethics statement
This study was carried out in accordance with the recommendations for the care and use of animals by the Office of Laboratory Animal Welfare (OLAW), NIH. The Institutional Animal Care and Use Committee (IACUC) of The University of North Carolina at Chapel Hill (UNC, Permit Number A-3410-01) approved the animal study protocol (IACUC #13-033) used in these studies.
Mice and in vivo infection
Female, 10-week-old, and 12-month-old BALB/cAnNHsD mice were ordered from Harlan Laboratories. Mouse infections were done as previously described20. Briefly, animals were brought into a BSL3 laboratory and allowed to acclimate for 1 week before infection. For infection and live-attenuated virus vaccination, mice were anesthetized with a mixture of ketamine and xylazine and infected intranasally, when challenged, with 50 μl of phosphate-buffered saline (PBS) or diluted virus with three or four mice per time point, per infection group per dose as described in the figure legends.
For individual mice, notations for infection including failure to inhale the entire dose, bubbling of inoculum from the nose, or infection through the mouth may have led to exclusion of mouse data at the discretion of the researcher; post-infection, no other pre-established exclusion or inclusion criteria are defined. No blinding was used in any animal experiments, and animals were not randomized. For vaccination, young and aged mice were vaccinated by footpad injection with a 20-μl volume of either 0.2 μg of double-inactivated SARS-CoV vaccine with alum or mock PBS; mice were then boosted with the same regimen 22 d later and challenged 21 d thereafter.
For all groups, as per protocol, animals were monitored daily for clinical signs of disease (hunching, ruffled fur, and reduced activity) for the duration of the experiment. Weight loss was monitored daily for the first 7 d, after which weight monitoring continued until the animals recovered to their initial starting weight or displayed weight gain continuously for 3 d.
All mice that lost greater than 20% of their starting body weight were ground-fed and further monitored multiple times per day as long as they were under the 20% cutoff. Mice that lost greater than 30% of their starting body weight were immediately sacrificed as per protocol. Any mouse deemed to be moribund or unlikely to recover was also humanely sacrificed at the discretion of the researcher. Euthanasia was performed using an isoflurane overdose and death was confirmed by cervical dislocation. All mouse studies were performed at the University of North Carolina (Animal Welfare Assurance #A3410-01) using protocols approved by the UNC Institutional Animal Care and Use Committee (IACUC).
Histological analysis.
The left lung was removed and submerged in 10% buffered formalin (Fisher) without inflation for 1 week. Tissues were embedded in paraffin and 5-μm sections were prepared by the UNC Lineberger Comprehensive Cancer Center histopathology core facility. To determine the extent of antigen staining, sections were stained for viral antigen using a commercially available polyclonal SARS-CoV anti-nucleocapsid antibody (Imgenex) and scored in a blinded manner by for staining of the airway and parenchyma as previously described20. Images were captured using an Olympus BX41 microscope with an Olympus DP71 camera.
Virus neutralization assays.
Plaque reduction neutralization titer assays were performed with previously characterized antibodies against SARS-CoV, as previously described11,
12,13
. Briefly, neutralizing antibodies or serum was serially diluted twofold and incubated with 100 p.f.u. of the different infectious clone, SARS-CoV strains for 1 h at 37 °C. The virus and antibodies were then added to a 6-well plate with 5 × 105 Vero E6 cells/well with multiple replicates (n ≥ 2). After a 1-h incubation at 37 °C, cells were overlaid with 3 ml of 0.8% agarose in a medium. Plates were incubated for 2 d at 37 °C, stained with neutral red for 3 h, and plaques were counted. The percentage of plaque reduction was calculated as (1 − (no. of plaques with an antibody/no. of plaques without antibody)) × 100.
Statistical analysis
All experiments were conducted contrasting two experimental groups (either two viruses, or vaccinated and unvaccinated cohorts). Therefore, significant differences in viral titer and histology scoring were determined by a two-tailed Student’s t-test at individual time points. Data were normally distributed in each group being compared and had similar variance.
Biosafety and biosecurity
Reported studies were initiated after the University of North Carolina Institutional Biosafety Committee approved the experimental protocol (Project Title: Generating infectious clones of bat SARS-like CoVs; Lab Safety Plan ID: 20145741; Schedule G ID: 12279).
These studies were initiated before the US Government Deliberative Process Research Funding Pause on Selected Gain-of-Function Research Involving Influenza, MERS, and SARS Viruses. This paper has been reviewed by the funding agency, the NIH. Continuation of these studies was requested, and this has been approved by the NIH.
SARS-CoV is a select agent. All work for these studies was performed with approved standard operating procedures (SOPs) and safety conditions for SARS-CoV, MERs-CoV, and other related CoVs. Our institutional CoV BSL3 facilities have been designed to conform to the safety requirements that are recommended in the Biosafety in Microbiological and Biomedical Laboratories (BMBL), the US Department of Health and Human Services, the Public Health Service, the Centers for Disease Control (CDC) and the NIH. Laboratory safety plans were submitted to, and the facility has been approved for use by, the UNC Department of Environmental Health and Safety (EHS) and the CDC. Electronic card access is required for entry into the facility.
All workers have been trained by EHS to safely use powered air-purifying respirators (PAPRs), and appropriate work habits in a BSL3 facility and active medical surveillance plans are in place. Our CoV BSL3 facilities contain redundant fans, emergency power to fans and biological safety cabinets and freezers, and our facilities can accommodate SealSafe mouse racks. Materials classified as BSL3 agents consist of SARS-CoV, bat CoV precursor strains, MERS-CoV and mutants derived from these pathogens. Within the BSL3 facilities, experimentation with an infectious virus is performed in a certified Class II Biosafety Cabinet (BSC).
All members of the staff wear scrubs, Tyvek suits and aprons, PAPRs and shoe covers, and their hands are double-gloved. BSL3 users are subject to a medical surveillance plan monitored by the University Employee Occupational Health Clinic (UEOHC), which includes a yearly physical, annual influenza vaccination and mandatory reporting of any symptoms associated with CoV infection during periods when working in the BSL3. All BSL3 users are trained in exposure management and reporting protocols, are prepared to self-quarantine and have been trained for safe delivery to a local infectious disease management department in an emergency situation. All potential exposure events are reported and investigated by EHS and UEOHC, with reports filed to both the CDC and the NIH.
Accession codes
Accessions
Protein Data Bank
2AJF
Change history
20 November 2015
In the version of this article initially published online, the authors omitted to acknowledge a funding source, USAID-EPT-PREDICT funding from EcoHealth Alliance, to Z.-L.S. The error has been corrected for the print, PDF and HTML versions of this article.
References
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Acknowledgments
Research in this manuscript was supported by grants from the National Institute of Allergy & Infectious Disease and the National Institute of Aging of the US National Institutes of Health (NIH) under awards U19AI109761 (R.S.B.), U19AI107810 (R.S.B.), AI085524 (W.A.M.), F32AI102561 (V.D.M.) and K99AG049092 (V.D.M.), and by the National Natural Science Foundation of China awards 81290341 (Z.-L.S.) and 31470260 (X.-Y.G.), and by USAID-EPT-PREDICT funding from EcoHealth Alliance (Z.-L.S.). Human airway epithelial cultures were supported by the National Institute of Diabetes and Digestive and Kidney Disease of the NIH under award NIH DK065988 (S.H.R.). We also thank M.T. Ferris (Dept. of Genetics, University of North Carolina) for the reviewing of statistical approaches and C.T. Tseng (Dept. of Microbiology and Immunology, University of Texas Medical Branch) for providing Calu-3 cells. Experiments with the full-length and chimeric SHC014 recombinant viruses were initiated and performed before the GOF research funding pause and have since been reviewed and approved for continued study by the NIH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Author information
Affiliations
Department of Epidemiology, the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Vineet D Menachery
, Boyd L Yount Jr
, Kari Debbink
, Lisa E Gralinski
, Jessica A Plante
, Rachel L Graham
, Trevor Scobey
, Eric F Donaldson
& Ralph S Baric Department of Microbiology and Immunology, the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Kari Debbink
& Ralph S Baric National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas, USA Sudhakar Agnihothram Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Xing-Yi Ge
& Zhengli-Li Shi Department of Cell Biology and Physiology, the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Scott H Randell Cystic Fibrosis Center, Marsico Lung Institute, the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Scott H Randell Institute for Research in Biomedicine, Bellinzona Institute of Microbiology, Zurich, Switzerland Antonio Lanzavecchia Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA Wayne A Marasco Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA Wayne A Marasco
Contributions
V.D.M. designed, coordinated and performed experiments, completed the analysis and wrote the manuscript. B.L.Y. designed the infectious clone and recovered chimeric viruses; S.A. completed neutralization assays; L.E.G. helped perform mouse experiments; T.S. and J.A.P. completed mouse experiments and plaque assays; X.-Y.G. performed pseudotyping experiments; K.D. generated structural figures and predictions; E.F.D. generated phylogenetic analysis; R.L.G. completed RNA analysis; S.H.R. provided primary HAE cultures; A.L. and W.A.M. provided critical monoclonal antibody reagents; and Z.-L.S. provided SHC014 spike sequences and plasmids. R.S.B. designed experiments and wrote the manuscript.
Corresponding authors
Correspondence to Vineet D Menachery or Ralph S Baric.
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Competing interests
The authors declare no competing financial interests.
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Menachery, V., Yount, B., Debbink, K. et al. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508–1513 (2015). https://doi.org/10.1038/nm.3985
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Received 12 June 2015
Accepted 08 October 2015
Published 09 November 2015
Issue Date December 2015
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veteranstoday--2020-03-23--4-Star General chosen to succeed Trump command in emergency

The reportedly approved sweeping contingency plans involve everything from the evacuation of civilian authorities and their families to a secure location , all the way to schemes to circumvent the guidelines for the transfer of power set forth in the US Constitution .
The Trump administration has drawn up secret standby orders to put the military in power in the event the civilian authorities are disabled by the coronavirus pandemic , a Newsweek report has it , outlining the details of the emergency plan in what ’ s called “ extraordinary circumstances ” .
More specifically , it is Terrence @ @ @ @ @ @ @ general and former fighter pilot who is reportedly the designated “ combatant commander ” .
So , what do we know about him so far ?
• He is the head of US Northern Command ( Northcom ) – a military authority in charge of homeland defence under the Continuity of Government Commission ’ s contingency plans .
• Terrence O ’ Shaughnessy also doubles as the chief of the North American Aerospace Defence Command , which has of late been frequently deployed by Trump to deal with illegal crossings at the Mexican-American @ @ @ @ @ @ @ domestic policies .
One of the latest examples – the large-scale Operation Faithful Patriot , from October-November 2018 .
• The Gen. was previously Deputy Commander at the United Nations Command Korea and led the US Pacific Air Forces Command , being in charge of joint operations in a region encompassing over half of the globe and as many as 36 nations .
• The senior general is a fighter pilot with 3,000 hours of flight time , including 168 combat hours in the F-16 Fighting Falcon .
Pentagon chief Mark Esper reportedly @ @ @ @ @ @ @ instructing Northcom to fulfil plans for a pandemic ( even before it was declared ) and urging it and East Coast military units to “ prepare to deploy ” for possible “ extraordinary ” missions , while not completely ruling out martial law .
Seven contingency plans are understood to have been approved , three of them overseeing missions to rescue and relocate President Trump , Vice President Mike Pence and their families , move the secretary of defence and other national security leaders , and congressional and court grandees to a secure @ @ @ @ @ @ @ maintained from a bunker in Maryland .
Another plan , codenamed Granite Shadow , envisages domestic ops involving weapons of mass destruction .
According to a military planner who spoke to the author of the Newsweek report , martial law is a “ pretty straightforward process ” that applies when one city or region has been devastated , “ but with coronavirus , where the effect is nationwide , we ’ re in territory we ’ ve never been in before ” , he concluded unambiguously .
The raging epidemic has been sweeping @ @ @ @ @ @ @ of cases in the US currently at 34,717 .
The tally indicates the country is the third-most affected in the world , after China and Italy .
The death toll in the US , meanwhile , is 452 , one of the lowest .
Trump ’ s inner circle , including POTUS himself , have so far tested negative for the novel viral infection , although a number of White House staffers self-isolated and have been working from home as a preventive anti-coronavirus measure .

The reportedly approved sweeping contingency plans involve everything from the evacuation of civilian authorities and their families to a secure location, all the way to schemes to circumvent the guidelines for the transfer of power set forth in the US Constitution.
The Trump administration has drawn up secret standby orders to put the military in power in the event the civilian authorities are disabled by the coronavirus pandemic, a Newsweek report has it, outlining the details of the emergency plan in what’s called “extraordinary circumstances”.
More specifically, it is Terrence O’Shaughnessy, a four-star 56-year-old general and former fighter pilot who is reportedly the designated “combatant commander”. So, what do we know about him so far?
He is the head of US Northern Command (Northcom) – a military authority in charge of homeland defence under the Continuity of Government Commission’s contingency plans.
Terrence O’Shaughnessy also doubles as the chief of the North American Aerospace Defence Command, which has of late been frequently deployed by Trump to deal with illegal crossings at the Mexican-American border – a central issue to his domestic policies. One of the latest examples – the large-scale Operation Faithful Patriot, from October-November 2018.
The Gen. was previously Deputy Commander at the United Nations Command Korea and led the US Pacific Air Forces Command, being in charge of joint operations in a region encompassing over half of the globe and as many as 36 nations.
The senior general is a fighter pilot with 3,000 hours of flight time, including 168 combat hours in the F-16 Fighting Falcon.
Pentagon chief Mark Esper reportedly signed standby orders on 1 February, instructing Northcom to fulfil plans for a pandemic (even before it was declared) and urging it and East Coast military units to “prepare to deploy” for possible “extraordinary” missions, while not completely ruling out martial law.
Seven contingency plans are understood to have been approved, three of them overseeing missions to rescue and relocate President Trump, Vice President Mike Pence and their families, move the secretary of defence and other national security leaders, and congressional and court grandees to a secure location.
Government operations will reportedly be maintained from a bunker in Maryland. Another plan, codenamed Granite Shadow, envisages domestic ops involving weapons of mass destruction.
According to a military planner who spoke to the author of the Newsweek report, martial law is a “pretty straightforward process” that applies when one city or region has been devastated, “but with coronavirus, where the effect is nationwide, we’re in territory we’ve never been in before”, he concluded unambiguously.
The raging epidemic has been sweeping across the globe, with the number of cases in the US currently at 34,717. The tally indicates the country is the third-most affected in the world, after China and Italy. The death toll in the US, meanwhile, is 452, one of the lowest.
Trump’s inner circle, including POTUS himself, have so far tested negative for the novel viral infection, although a number of White House staffers self-isolated and have been working from home as a preventive anti-coronavirus measure.


veteranstoday--2020-03-23--Chloroquine Is A Very, Very High Risk Treatment For COVID-19

Health Editor ’ s Note : Trump announced that patients with pneumonia who are infected with COVID-19 were going to be treated with chloroquine which is a commonly used anti-malarial drug .
He made another mistake that could be deadly to people ( mostly males of African and Mediterranean descent ) who have an often hidden genetic defect that affects important enzyme functions .
The G6PD gene is responsible for processing of carbohydrate and protection of cells from harmful effects of free oxygen radicals .
To make an important story shorter , someone with the G6PD gene , who is @ @ @ @ @ @ @ the red blood cells which are responsible for caring oxygen to the body tissues .
Red blood cells rupturing is a major assault on a body , let alone having COVID-19 as well .
Why the concern for a genetic deficiency that many have not heard of ?
G6PD gene deficiency is common with 1 in 10 African-American U.S. males having this defect .
This is the second most common human enzyme defect which affects 400 million people throughout the world .
Did Trump misspeak as he so often does ?
These are @ @ @ @ @ @ @ since he has no medical background would seem like Greek to him .
Did he mean Hydroxychloroquine ?
Hydroxychloroquine is a drug which is slightly different in molecular structure from chloroquine thus it does not cause bursting of red blood cells in people with G6PD .
This drug has shown effectiveness against COVID-19 and can be given to those who may or do have G6PD deficiency .
The Chinese have done a study on both chloroquine and hydroxychloroquine and found that hydroxychloroquine is the most effective in treating COVID-19 .
It seems to @ @ @ @ @ @ @ should be used…..Carol Chloroquine Is Not a Harmless Panacea for COVID-19 by Dan J. Vick MD , DHA , MBA , CPE/a pathologist and former hospital executive , is a member of the graduate teaching faculty in the Master of Health Administration Program , School of Health Sciences , in the Herbert H. & Grace A. Dow College of Health Professions at Central Michigan University in Mount Pleasant .
Printed in MedPage Today .
During a March 19 press conference , President Trump announced that chloroquine , a drug long used to treat @ @ @ @ @ @ @ available for those stricken with COVID-19 .
This followed news of preliminary research , including a limited study in Australia , in which chloroquine showed promise in eradicating the coronavirus in some patients .
Chinese research published in February suggested efficacy and safety of chloroquine in treating pneumonia associated with COVID-19 .
A subsequent Chinese study involving the use of both chloroquine and its molecular cousin , hydroxychloroquine , determined that hydroxychloroquine is the more potent of these two drugs in its inhibition of this novel coronavirus .
Controversy enveloped the announcement by @ @ @ @ @ @ @ that chloroquine has already been approved for this use by the FDA .
The agency ’ s commissioner , Stephen Hahn , MD , quickly clarified that larger studies still need to be conducted to determine the safety and effectiveness of chloroquine for treating COVID-19 .
Much of the discussion that continued in the media throughout the day concerned whether the drug will truly prove effective and , if so , how long will it be until this medication gets the green light for patient use .
There was also confusion as to @ @ @ @ @ @ @ reports , and the president himself , made mention of hydroxychloroquine as well .
The distinction is important , as will be seen shortly .
While others are focusing on the drug effectiveness component in this debate , I am more concerned about the safety with regard to chloroquine .
What I haven ’ t seen mentioned is the contraindication for use of the drug in people who have glucose-6-phosphate dehydrogenase ( G6PD ) deficiency .
For those who need a refresher from medical school , G6PD deficiency is an X-linked recessive genetic @ @ @ @ @ @ @ in males .
It is found predominantly in people of African or Mediterranean origin .
As a result of mutations in the G6PD gene , the amount of G6PD is either reduced or its structure is significantly altered so that it can not perform its usual enzymatic functions .
G6PD has two primary functions : it plays a role in carbohydrate processing and , important for this discussion , it helps protect cells from the harmful effects of free oxygen radicals .
These reactive oxygen molecules are byproducts of normal cell function .
@ @ @ @ @ @ @ known as the pentose phosphate pathway and produces another molecule called NADPH .
The latter has a direct role in ridding cells of the free oxygen radicals before they build up to toxic levels .
This function is especially essential for red blood cells , which , unlike other cells in the body , lack additional NADPH-producing enzymes .
In the presence of a buildup of reactive oxygen species , red blood cells are prematurely destroyed , causing a condition called hemolytic anemia .
This can result in jaundice , shortness of breath @ @ @ @ @ @ @ the remaining red blood cells ) , and tachycardia .
In severe cases , it can cause acute kidney failure and death .
However , many people with G6PD deficiency are asymptomatic and not aware that they have it until something triggers an episode of hemolytic anemia .
Common triggers include bacterial or viral infections and treatment with certain drugs .
One drug commonly associated with hemolytic anemia in G6PD deficiency is … chloroquine .
Should this be a concern in the present debate over treating COVID-19 patients ?
In my opinion , @ @ @ @ @ @ @ ; in fact , it is the second most common human enzyme defect , affecting some 400 million people worldwide .
It affects 1 in 10 African-American males in the U.S .
It is common enough that it was written into an episode of the long-running TV series M*A*S*H. In this episode , the character Corporal Klinger , who was of Mediterranean descent , became seriously ill after being given an anti-malarial drug .
He was found to have hemolytic anemia and an association was made with the drug .
The ending credits @ @ @ @ @ @ @ .
Given the challenges of knowing who may or may not have G6PD deficiency , it would seem prudent not to use chloroquine to treat COVID-19 patients who may be at risk for this genetic condition .
The last thing they need is to have a serious respiratory disease compounded by hemolytic anemia , resulting in further loss of oxygenation .
Hydroxychloroquine , on the other hand , does not induce hemolytic anemia in people with G6PD deficiency despite the molecular similarity to chloroquine .
It has shown effectiveness in inhibiting the pandemic @ @ @ @ @ @ @ this is the drug to which the president and Hahn were referring .
Hydroxychloroquine is where the FDA should direct its testing efforts , and quickly , to determine whether this may be the silver bullet for treating COVID-19 .

Health Editor’s Note: Trump announced that patients with pneumonia who are infected with COVID-19 were going to be treated with chloroquine which is a commonly used anti-malarial drug. He made another mistake that could be deadly to people (mostly males of African and Mediterranean descent) who have an often hidden genetic defect that affects important enzyme functions. The G6PD gene is responsible for processing of carbohydrate and protection of cells from harmful effects of free oxygen radicals. To make an important story shorter, someone with the G6PD gene, who is given chloroquine, may experience rupture of the red blood cells which are responsible for caring oxygen to the body tissues. Red blood cells rupturing is a major assault on a body, let alone having COVID-19 as well.
Why the concern for a genetic deficiency that many have not heard of? G6PD gene deficiency is common with 1 in 10 African-American U.S. males having this defect. This is the second most common human enzyme defect which affects 400 million people throughout the world.
Did Trump misspeak as he so often does? These are drugs with well, drug names and since he has no medical background would seem like Greek to him. Did he mean Hydroxychloroquine? Hydroxychloroquine is a drug which is slightly different in molecular structure from chloroquine thus it does not cause bursting of red blood cells in people with G6PD. This drug has shown effectiveness against COVID-19 and can be given to those who may or do have G6PD deficiency. The Chinese have done a study on both chloroquine and hydroxychloroquine and found that hydroxychloroquine is the most effective in treating COVID-19. It seems to me that hydroxychloroquine is the drug that should be used…..Carol
Chloroquine Is Not a Harmless Panacea for COVID-19
by Dan J. Vick MD, DHA, MBA, CPE/a pathologist and former hospital executive, is a member of the graduate teaching faculty in the Master of Health Administration Program, School of Health Sciences, in the Herbert H. & Grace A. Dow College of Health Professions at Central Michigan University in Mount Pleasant. Printed in MedPage Today. During a March 19 press conference, President Trump announced that chloroquine, a drug long used to treat malaria, was going to be made available for those stricken with COVID-19. This followed news of preliminary research, including a limited study in Australia, in which chloroquine showed promise in eradicating the coronavirus in some patients. Chinese research published in February suggested efficacy and safety of chloroquine in treating pneumonia associated with COVID-19. A subsequent Chinese study involving the use of both chloroquine and its molecular cousin, hydroxychloroquine, determined that hydroxychloroquine is the more potent of these two drugs in its inhibition of this novel coronavirus. Controversy enveloped the announcement by the president, especially after he indicated that chloroquine has already been approved for this use by the FDA. The agency’s commissioner, Stephen Hahn, MD, quickly clarified that larger studies still need to be conducted to determine the safety and effectiveness of chloroquine for treating COVID-19. Much of the discussion that continued in the media throughout the day concerned whether the drug will truly prove effective and, if so, how long will it be until this medication gets the green light for patient use….Read More:
Carol graduated from Riverside White Cross School of Nursing in Columbus, Ohio and received her diploma as a registered nurse. She attended Bowling Green State University where she received a Bachelor of Arts Degree in History and Literature. She attended the University of Toledo, College of Nursing, and received a Master’s of Nursing Science Degree as an Educator. She has traveled extensively, is a photographer, and writes on medical issues. Carol has three children RJ, Katherine, and Stephen – one daughter-in-law; Katie – two granddaughters; Isabella Marianna and Zoe Olivia – and one grandson, Alexander Paul. She also shares her life with her husband Gordon Duff, many cats, and two rescues. Carol’s Archives 2009-2013


veteranstoday--2020-03-23--China and Russia help COVID West while under its sanctions

With united Europe MIA in its Covid-19 response , worst-hit nations turn to ‘ evil ’ Russia & China for help …by Damian Wilson , a UK journalist , ex-Fleet Street editor , financial industry consultant and political communications special advisor in the UK and EU .
[ Editor ’ s Note : This is a great critique of the EU failure , in the wake of the US failure , about being ready for and dealing with the coronavirus .
What an ultimate irony to have EU sanctioned Russia sharing its own Covid19 medical supplies with EU countries worst @ @ @ @ @ @ @ his expected demonization of China for the virus , his campaign defense plan for steering attention away from its tremendous success at closing Covid-19 down inside China .
The new low infection counts being published are from travelers being picked up incoming at airports and returning Chinese students .
Russia took early action to blunt Covid in the front end , so now has the luxury of helping those countries who have been diplomatically civil to Russia while the EU has it under sanction for the pretend violation of “ taking over ” @ @ @ @ @ @ @ Ukrainian MPs were threatening from the Rada that the Ukrainian nationalists were going to steal everything the Crimean Russians had and throw them out .
What did NATO do to help the EU in this situation ?
Nothing that we can see .
Is there an equivalent to the Army Corps of Engineers that is beginning to convert empty hotels into ICU compatible medical facilities over the next four weeks ?
In fact , it seems the EU pandemic preparation was as anemic as Trump ’ s , who is hard to beat @ @ @ @ @ @ @ he then tosses onto someone else for the blame .
All that was on Trump ’ s mind was not losing votes for this re-election .
He did not go deer in the headlights .
He was all in on an impossible gamble to hold the markets up so he could crow about all the wealth creation he had done .
When all is said and done that might turn out to have been a classic NeoCon pump and dump , with Trump in on it or lined up to take the fall @ @ @ @ @ @ @ has Pompeo the Pompous preened up and ready to go as a replacements as no one with half a brain has any confidence in Pence .
The Russian and Chinese people have a lot to be thankful for , and can be proud about how they fought the virus tooth and nail for all the world to see , can now watch as China , and neighbor Russia extend their hand to those who have bitten them… Jim W. Dean ] Jim 's Editor ’ s Notes are solely crowdfunded via PayPal Jim @ @ @ @ @ @ @ , Heritage TV Legacy archiving & more .
Thanks for helping .
Click to donate > > Brussels has all but abandoned them to their fates , so is it any wonder that Italy , Spain and other European countries have turned to Moscow and Beijing for meaningful support in terms of expert advice and medical supplies ?
When this coronavirus pandemic has passed and the nations of the European Union are counting the cost of decimated populations , shattered public health infrastructures and economic meltdowns , one thing is certain : the @ @ @ @ @ @ @ forever .
Before the global outbreak of this killer virus , Russia and China were always the West ’ s undisputed bogeymen .
Troublemakers , always up to mischief , playing dangerous geopolitical games intended to undermine Western democracies and divide allies in the EU and NATO .
Many in the Western mainstream media are addicted to a daily fix of either Russian or Chinese conspiracy , so they probably don ’ t appreciate the irony here .
Because things have changed .
In a big way .
It ’ s largely due @ @ @ @ @ @ @ tricksy ” outsider superpowers that some European nations , fully paid-up , long-term members of the EU , have even a remote chance of getting to grips with the immense public health demands caused by the outbreak of the coronavirus .
And thank heavens for that , because if either Italy or Spain had been looking to the EU — or the USA for that matter — for real , on-the-ground assistance that would enable them to simply stop people dying in their thousands in overcrowded hospitals , then they would have found @ @ @ @ @ @ @ the ECB weighed in only last week , with an unprecedented fiscal bazooka aimed at reducing the economic impact that is being caused by the virus ( ar at least by the measures governments are taking to try to stop it ) .
But that ’ s fighting the virus in one manner only , very much after the fact and certainly not on the frontline .
This significance of that fiscal help will only really be apparent once the health crisis is over and the full , disastrous economic consequences are realized @ @ @ @ @ @ @ far from over right now is what needs immediate attention , like providing more medical staff , personal protection gear , ventilators , hospitals and , obviously , a vaccine .
But the message from Brussels to Italy , Spain and other EU members is clear : in dealing with the pandemic and the thousands of dead citizens it leaves in its wake , you are on your own .
The EU ’ s abject hopelessness and indifference started to become clear some weeks back in the face of the escalating virus , @ @ @ @ @ @ @ facemasks and medical equipment was banned by some EU members , including Germany .
It ’ s unlikely the Italians will forget that .
Compare it with the generosity of the Chinese consumer electronics company Xiaomi which has sent tens of thousands of face masks to Italy at the same time as Beijing has filled planes with medical experts and crucial equipment of which Rome was , and remains , in desperate need .
Compare it too with the help from Moscow sent over the weekend : 100 virus specialists , disinfection trucks @ @ @ @ @ @ @ at the request of Italy .
It seems the close ties the Italian government maintained with Russia over the last few years , particularly at the initiative of former coalition foreign minister Matteo Salvini , have paid off .
His refusal to be drawn into the EU ’ s Kremlin-bashing of recent times , preferring to cultivate a closer friendship of his own , has to be applauded for the dividends it has produced in the Italian hour of need .
And one unintended consequence of that must be that it is driving @ @ @ @ @ @ @ nuts .
It also won ’ t escape the attention of NATO that the aid from Russia is coming via military channels , which must sting those of its members with a more hawkish view towards Moscow .
Just so they don ’ t miss the irony , the supplies bear a “ From Russia with Love ” label .
What can not be denied is that actions speak louder than words .
The people of those nations being helped from outside the EU will be eternally grateful for that assistance .
It @ @ @ @ @ @ @ .
It ’ s brigades of medical experts , boxes of supplies delivered in big planes .
It will save the lives of family and friends and it will provide help on the frontline where it is most needed .
In Verduno , near Turin in northern Italy , a hospital that has been under construction for 20 years , and is still unfinished , will open as a Covid-19 hospital next week , and will take in its first 60 coronavirus victims .
In comparison , the Italian government will be well @ @ @ @ @ @ @ working around the clock , built a 1000-bed hospital in just 10 days , and that Russia , with parallel ambition , is looking to finish a virus hospital on the outskirts of Moscow within a month .
It is these sorts of highly visible acts that people need to see .
It builds confidence from despair and hope from misery .
Confidence and hope .
As millions of terrified Europeans sit confined in their homes today , isolated voluntarily or by law , those are two things in critically short supply .

With united Europe MIA in its Covid-19 response, worst-hit nations turn to ‘evil’ Russia & China for help
…from Russia Today, Moscow
…by Damian Wilson, a UK journalist, ex-Fleet Street editor, financial industry consultant and political communications special advisor in the UK and EU.
[ Editor’s Note: This is a great critique of the EU failure, in the wake of the US failure, about being ready for and dealing with the coronavirus. What an ultimate irony to have EU sanctioned Russia sharing its own Covid19 medical supplies with EU countries worst hit.
Meanwhile, Trump was doing his expected demonization of China for the virus, his campaign defense plan for steering attention away from its tremendous success at closing Covid-19 down inside China. The new low infection counts being published are from travelers being picked up incoming at airports and returning Chinese students.
Russia took early action to blunt Covid in the front end, so now has the luxury of helping those countries who have been diplomatically civil to Russia while the EU has it under sanction for the pretend violation of “taking over” Crimea with its vastly Russian population when Ukrainian MPs were threatening from the Rada that the Ukrainian nationalists were going to steal everything the Crimean Russians had and throw them out.
What did NATO do to help the EU in this situation? Nothing that we can see. Is there an equivalent to the Army Corps of Engineers that is beginning to convert empty hotels into ICU compatible medical facilities over the next four weeks?
In fact, it seems the EU pandemic preparation was as anemic as Trump’s, who is hard to beat when it comes to screws ups that he then tosses onto someone else for the blame.
All that was on Trump’s mind was not losing votes for this re-election. He did not go deer in the headlights. He was all in on an impossible gamble to hold the markets up so he could crow about all the wealth creation he had done.
When all is said and done that might turn out to have been a classic NeoCon pump and dump, with Trump in on it or lined up to take the fall for it. The Deep State already has Pompeo the Pompous preened up and ready to go as a replacements as no one with half a brain has any confidence in Pence.
The Russian and Chinese people have a lot to be thankful for, and can be proud about how they fought the virus tooth and nail for all the world to see, can now watch as China, and neighbor Russia extend their hand to those who have bitten them… Jim W. Dean ]
Jim's Editor’s Notes are solely crowdfunded via PayPal
Jim's work includes research, field trips, Heritage TV Legacy archiving & more. Thanks for helping. Click to donate >>
– First published … March 23, 2020 –
Brussels has all but abandoned them to their fates, so is it any wonder that Italy, Spain and other European countries have turned to Moscow and Beijing for meaningful support in terms of expert advice and medical supplies?
When this coronavirus pandemic has passed and the nations of the European Union are counting the cost of decimated populations, shattered public health infrastructures and economic meltdowns, one thing is certain: the dynamics within the bloc will have changed forever.
Before the global outbreak of this killer virus, Russia and China were always the West’s undisputed bogeymen. Troublemakers, always up to mischief, playing dangerous geopolitical games intended to undermine Western democracies and divide allies in the EU and NATO.
Many in the Western mainstream media are addicted to a daily fix of either Russian or Chinese conspiracy, so they probably don’t appreciate the irony here. Because things have changed. In a big way.
It’s largely due to the generosity of these two “tricksy” outsider superpowers that some European nations, fully paid-up, long-term members of the EU, have even a remote chance of getting to grips with the immense public health demands caused by the outbreak of the coronavirus.
And thank heavens for that, because if either Italy or Spain had been looking to the EU — or the USA for that matter — for real, on-the-ground assistance that would enable them to simply stop people dying in their thousands in overcrowded hospitals, then they would have found themselves waiting in vain.
Sure, the ECB weighed in only last week, with an unprecedented fiscal bazooka aimed at reducing the economic impact that is being caused by the virus (ar at least by the measures governments are taking to try to stop it). But that’s fighting the virus in one manner only, very much after the fact and certainly not on the frontline.
This significance of that fiscal help will only really be apparent once the health crisis is over and the full, disastrous economic consequences are realized. And the fact that it is far from over right now is what needs immediate attention, like providing more medical staff, personal protection gear, ventilators, hospitals and, obviously, a vaccine.
But the message from Brussels to Italy, Spain and other EU members is clear: in dealing with the pandemic and the thousands of dead citizens it leaves in its wake, you are on your own.
The EU’s abject hopelessness and indifference started to become clear some weeks back in the face of the escalating virus, when the export beyond national borders of facemasks and medical equipment was banned by some EU members, including Germany.
It’s unlikely the Italians will forget that. Compare it with the generosity of the Chinese consumer electronics company Xiaomi which has sent tens of thousands of face masks to Italy at the same time as Beijing has filled planes with medical experts and crucial equipment of which Rome was, and remains, in desperate need.
Compare it too with the help from Moscow sent over the weekend: 100 virus specialists, disinfection trucks and medical equipment. All being sent at the request of Italy.
It seems the close ties the Italian government maintained with Russia over the last few years, particularly at the initiative of former coalition foreign minister Matteo Salvini, have paid off. His refusal to be drawn into the EU’s Kremlin-bashing of recent times, preferring to cultivate a closer friendship of his own, has to be applauded for the dividends it has produced in the Italian hour of need.
And one unintended consequence of that must be that it is driving critics of the controversial Italian populist absolutely nuts.
It also won’t escape the attention of NATO that the aid from Russia is coming via military channels, which must sting those of its members with a more hawkish view towards Moscow. Just so they don’t miss the irony, the supplies bear a “From Russia with Love” label.
What cannot be denied is that actions speak louder than words. The people of those nations being helped from outside the EU will be eternally grateful for that assistance. It’s help that can be seen. It’s brigades of medical experts, boxes of supplies delivered in big planes. It will save the lives of family and friends and it will provide help on the frontline where it is most needed.
In Verduno, near Turin in northern Italy, a hospital that has been under construction for 20 years, and is still unfinished, will open as a Covid-19 hospital next week, and will take in its first 60 coronavirus victims.
In comparison, the Italian government will be well aware that China, using 7,000 builders working around the clock, built a 1000-bed hospital in just 10 days, and that Russia, with parallel ambition, is looking to finish a virus hospital on the outskirts of Moscow within a month.
It is these sorts of highly visible acts that people need to see. It builds confidence from despair and hope from misery.
Confidence and hope. As millions of terrified Europeans sit confined in their homes today, isolated voluntarily or by law, those are two things in critically short supply.


veteranstoday--2020-03-23--Free: Online Culture, History, Science Collections

Carol graduated from Riverside White Cross School of Nursing in Columbus , Ohio and received her diploma as a registered @ @ @ @ @ Green State University where she received a Bachelor of Arts Degree in History and Literature @ @ @ @ @ of Toledo , College of Nursing , and received a Master ’ s of Nursing @ @ @ @ @ has traveled extensively , is a photographer , and writes on medical issues .
Carol @ @ @ @ @ Katherine , and Stephen – two daughters-in-law ; Suzy and Katie – two granddaughters ; @ @ @ @ @ – and one grandson , Alexander Paul .
She also shares her life with husband @ @ @ @ @ , two rescue pups , and two guinea pigs .

Carol graduated from Riverside White Cross School of Nursing in Columbus, Ohio and received her diploma as a registered nurse. She attended Bowling Green State University where she received a Bachelor of Arts Degree in History and Literature. She attended the University of Toledo, College of Nursing, and received a Master’s of Nursing Science Degree as an Educator.
She has traveled extensively, is a photographer, and writes on medical issues. Carol has three children RJ, Katherine, and Stephen – one daughter-in-law; Katie – two granddaughters; Isabella Marianna and Zoe Olivia – and one grandson, Alexander Paul. She also shares her life with her husband Gordon Duff, many cats, and two rescues.