How Boris Survived

Readers will not be taken aback to learn that I was surprised that PM Boris Johnson survived his brush with Covid-19 .
I knew that he was desperately ill and had been on a ventilator .
I assumed , since he was in an NHS hospital , that he had been written off , along with all seriously ill NHS Covid-19 patients , and was being given palliative care only .
It is now increasingly clear that he was treated with the anti-viral drug Remdesivir .
If that is right the political consequences will be huge .
Thousands of NHS @ @ @ @ @ @ @ have died .
Once that gets out Boris is finished , no offense intended .
The NHS won ’ t be far behind .
The General Medical Council were right to oppose the creation of the NHS .
As they foresaw way back in 1948 socialised medicine would lead , inevitably , to decisions being taken by bureaucrats rather than doctors , in the interests of the state rather than the patient .
In practice key decisions on NHS strategy in the Covid-19 outbreak have been taken by the Cabinet Office , which @ @ @ @ @ @ @ Sending the PM to an NHS hospital looked like a death sentence .
On Cabinet Office orders the only drugs known to be effective in combatting Covid-19 , Hydroxychloriquine and Remdesivir , have been withheld from NHS patients .
At the same time government ( in reality , Cabinet Office ) policy , to which I am strongly opposed , is to sacrifice frontline NHS staff by withholding information that the virus is airborne .
They ’ ve been going down like ninepins .
It didn ’ t occur to me that the @ @ @ @ @ @ @ PM only .
No offense to the PM , but far more important people , such as the actor Tim Brooke-Taylor , have been allowed to die without receiving proper treatment .
Prime Ministers are important of course , but even the PM ’ s strongest supporters would scarcely suggest that he was worth a Goodie !
( The Goodies was an hilarious 1970s comedy show . )
Boris can be replaced .
Tim Brooke-Taylor can ’ t .
The official British government line is that neither Hydroxychloriquine and Remdesivir have been proved @ @ @ @ @ @ @ is nonsense .
The Marseilles trial I ’ ve been writing about was peer-reviewed and published in a reputable journal .
It may have been small-scale but it was conducted properly .
The drugs have been used to good effect in the US , China and now Britain .
There is no need to hold up their deployment any longer .
It ’ s perfectly clear that Hydroxychloriquine is safer than Chloriquine and that even Hydroxychloriquine is not suitable for all patients , for example those with heart defects .
It ’ s @ @ @ @ @ @ @ for the majority of Covid-19 patients , particularly in the early stages of hospital admission .
Remdesivir seems to be more suitable for patients in acute respiratory distress .
Bureaucrats holding up the prescription of both drugs are playing with lives .
When was the President told ?
We know that Covid-19 is a bioweapon , that it was released , unintentionally , from the Wuhan Institute of Virology in mid-November and that Peking covered it up for weeks , whilst tens of thousands of their people died .
We also know that @ @ @ @ @ @ @ It ’ s also clear that the weapon was mainly designed to cause economic damage , not deaths .
CIA , who as revealed on this site were involved in the Obama-era Covid-19 program , working with the ChiComs , clearly knew in November .
Equally clearly the White House were not told .
It seems that the President wasn ’ t told until around the middle of January , around about the same time Covid-19 started to take off in the States .
It was also the time of maximum distraction over @ @ @ @ @ @ @ is being strongly criticised by the Democrats for delay , given the circumstances he moved quite quickly .
Insanely , the British government still hasn ’ t banned flights from China !
The mere fact the CIA knew that something was up in Wuhan most definitely does not mean that the White House knew .
The Agency are strongly backing Joe Biden in November and were in the Russia and Ukraine hoaxes up to their necks .
Almost certainly Langley thought that Covid-19 would be an ideal way to embarrass the President and @ @ @ @ @ @ @ The fact that tens of thousands of Americans might die in the process doesn ’ t seem to have troubled them .
The CIA are well used to writing off American lives .
It is not unreasonable to suppose that senior Democrats were briefed in by the CIA and were prepared to go along with the plan to keep the President in the dark .
Are the Democrats that cynical ?
You bet .
This is the party that knowingly installed a non-citizen in the White House and then covered it up .
@ @ @ @ @ @ @ went along with an absurd conspiracy theory that Russia somehow hacked into the party ’ s server , when the download speed ruled hacking out completely , and when that nonsense fell apart they invented another hoax concerning the Ukraine .
It ’ s also the party which cynically relies upon African-American voters not being willing to go elsewhere and plans to throw them under a bus by giving their jobs to Hispanic immigrants .
As that nice man Ted Hayes , an African-American activist in LA , explained to me when taking @ @ @ @ @ @ @ years ago , it ’ s African-Americans who mostly suffer from illegal Hispanic immigration .
Middle-class whites tend to benefit if anything , as they get cheap domestic labor .
And , yes , I ’ ve been to Skid Row , where as a white upper middle class English barrister I was received with great civility and good humor .
You tend to meet nicer people there than in Hollywood .
Of course the Democrats are prepared to throw working-class whites under a bus as well , by taking their votes in @ @ @ @ @ @ @ jobs to China .
They ’ re so desperate to win in November that they ’ re even prepared to indulge in large-scale voter fraud .
( Then they scream blue murder when the Administration takes sensible measures to prevent it . )
I have little doubt that senior figures in the Democratic Party were alerted to the Wuhan outbreak by the CIA as far back as November and planned to take political advantage of it .
I suspect they also knew that the Agency , CDC and DNI had all agreed to @ @ @ @ @ @ @ President .
CDC have about a dozen people embedded in the evil , genocidal World Health Organisation , no offense intended , and were in the loop by December at the latest .
President Trump was absolutely right , with respect , to withhold funding from WHO .
In so doing the President has shown real leadership .
Other Western countries should follow America ’ s example .
It ’ s high time that the UN and its agencies were given a real smack .
WHO have not only covered up China ’ @ @ @ @ @ @ @ CIA assistance of course ) , but her role in creating SARS and MERS as well .
Working with CDC in Atlanta they have also covered up Germany ’ s role in creating the Spanish Flu bio-weapon .
WHO ’ s shameful actions have cost many lives .
Bio-weapons typically are airborne , because they are genetically engineered for easy transmission .
Hiding their origin not only protects those states which develop them , but puts medical personnel at risk .
The final death toll from WHO ’ s actions is likely to @ @ @ @ @ @ @ The Beatles and the Supreme Court The Beatles are back in the news .
Not the long-haired lovers from Liverpool but the murderous ISIS terrorists who liked to chop people ’ s heads off .
Maha Elgizouli , the mother of one of these clowns , has outrageously secured a with respect ludicrous decision from the UK Supreme Court effectively preventing British witnesses from giving evidence at the trial of her son , Shafee El Sheikh .
( Elgizouli v. Secretary of State for the Home Department [ 2020 ] UKSC 10 ) @ @ @ @ @ @ @ ’ s nothing much ‘ alleged ’ about El Sheikh ’ s role as an ISIS executioner .
His supporters aren ’ t seriously arguing that it ’ s a case of mistaken identity .
Their argument is that the Sudanese terrorist should be given a soft sentence , presumably with a view to encouraging other young Islamists to go around chopping people ’ s heads off .
As a result of this decision it ’ s now unlikely that you guys will be able to prosecute .
The terrorist-sympathising Crown Prosecution Service @ @ @ @ @ @ @ that he can not be prosecuted in the UK .
Since he ’ s no longer a British Citizen he has no right of entry into the UK in any event .
He won ’ t be coming back here .
It looks like he ’ ll be headed to Gitmo .
One option of course would be to hand him back to the Syrian Democratic Forces .
They would have jurisdiction , since they captured him on the battlefield .
They could give him a fair summary trial by court martial and @ @ @ @ @ @ @ ( No need to chop his head off . )
Since the Beatles believed in videoing their executions there ’ s no particular reason why the SDF couldn ’ t broadcast the execution on YouTube , subject of course to any rules YouTube may have about broadcasting live executions .
Another option would be to amend the UK Data Protection Act and make it clear that personal data can be transmitted to states with which Her Majesty enjoys diplomatic relations for law enforcement purposes regardless of that state ’ s sentencing policies .
@ @ @ @ @ @ @ ’ s finding that the Data Protection Act was somehow breached by sending witness statements and other material to the Justice Department .
The reasoning , if reasoning is not too strong a word with respect , of the justices is unlikely to stand the test of time .
Their interpretation of the Data Protection Act is strained to say the least .
Lord Kerr in particular strongly disagreed with the softening of the British government ’ s opposition to the death penalty , banging on about the policy of previous governments as @ @ @ @ @ @ @ with respect , may have preferred the policy of the Blair and Brown governments , but they ’ re out of office .
Lord Kerr , a half-crazed Euronutter , no offense intended , also made the hugely controversial comment , likely to be the court ’ s death-knell , that now that we ’ re out of the EU the judges can bring back community law by the side-wind of declaring it to be part of the Common Law , against the will of the people and Parliament .
With the greatest @ @ @ @ @ @ @ right to remove Lord Kerr from office and abolish the court .
We should go back to having the House of Lords .
The House of Lords , with respect , was not immune from handing down silly decisions , Factortame and Metric Martyrs being cases in point , but none of them was this crazy .
Basically what the Supreme Court has said is that it ’ s OK to go around chopping innocent people ’ s heads off .
Maybe their perspective might change if one of the justices were to @ @ @ @ @ @ @ updated , with an additional chapter on the F-111K by Tony Buttler , this is the most comprehensive work ever published on one of Britain ’ s greatest ever technological achievements , the superb British Aircraft Corporation TSR-2 .
Tim , sadly , died in 2015 .
TSR-2 is a technical tour de force , giving a detailed description of the aircraft ’ s specification and advanced systems .
There are excellent line drawings and fine studies of the prototype , XR219 , flying from Boscombe Down and Wharton .
Most of those @ @ @ @ @ @ @ s brilliant chief test pilot , ‘ Bee ’ Beamont , a friend of a friend , who did superb work in ‘ Tiffies ’ , strafing Jerry after D-Day .
Sadly however the book is let down by the failure to consider the intelligence aspects .
Both the Air Minister , Roy ‘ von ’ Jenkins , and the Prime Minister , Harold ‘ von ’ Wilson , who essentially took the fateful decision to cancel the project between them , were German agents .
The Defence Minister , Denis Healey ( @ @ @ @ @ @ @ Party conference ) unfairly took most of the blame .
Denis was a nice chap , but he was a house-trained idiot , no offense intended , who swallowed the Treasury ’ s line that we couldn ’ t afford the aircraft .
As I argue in Spyhunter Whitehall never intended the TSR-2 to go into production , any more than they really intended to buy the F-111 .
The intent all along was to concentrate resources on one combat aircraft , cancel it , smash up the UK ’ s aerospace sector @ @ @ @ @ @ @ Union .
The plan worked , sadly .
‘ Von ’ Wilson ’ s talk of the “ white heat of the scientific revolution ” was just that – talk .
Wilson wanted to deskill the UK and hold back our technological development .
TSR-2 ’ s replacement was a batch of old Blackburn Buccaneers transferred from the Navy after Labour ’ s equally disastrous decision to cancel the CVA01 and CVA02 attack carriers .
Fine aircraft that it was , when first used , by the South Africans , to drop a @ @ @ @ @ @ @ blown out of the sky by the shock-wave .
There is no doubt that a TSR-2 would have survived the delivery of that particular nuclear payload .
It was capable of a staggering Mach 1.2 at 250 feet and could have egressed at Mach 2.2 at altitude having delivered a nuclear strike package .
The aircraft not only met its hugely demanding specification , it exceeded it .
It could take off from an unprepared strip no more than 3,000 long , fly more than 1,000 nautical miles , accurately deliver a nuclear @ @ @ @ @ @ @ profile , with supersonic approach to the target , and fly 1,000 nautical miles back without inflight refuelling .
Its nav-attack and recon systems , including inertial navigation , line-scan camera and sidescan radar , were the most advanced of any aircraft in 1964 .
When flying asymmetric , on just one of her mighty Bristol Siddeley Olympus 22-R engines Bee managed to pull away from Jimmy Dell in the English Electric Lightning T4 chase plane .
The finest interceptor in the world in the mid-60s , the Lightning was a Mach 2-capable @ @ @ @ @ @ @ 50,000 feet a minute .
Arguably no other aircraft in existence could pull away from a Lightning on just 50 % of combat power .
Staggeringly , the TSR-2 could also climb at 50,000 feet a minute , clean , with no external stores or tanks .
That ’ s nearly a 1,000 feet a second , for a bomber .
McClelland gives the lie to the claim that the TSR-2 lacked maneuverability .
Its wing-loading was less than the F-111 ’ s , which was nominally a fighter , hence the ‘ @ @ @ @ @ @ @ TSR-2 could out-turn most fighters of the era .
It handled superbly .
Interestingly , McClelland reveals that the aircraft had provision for an arrester hook .
Its heavy-duty undercarriage could have coped with carrier landings and it was stressed for catapult launch , indeed it was a very strong aircraft , designed as it was to fly supersonic close to the ground , using terrain-following radar .
Were the Navy at one time thinking of TSR-2 as a Buccaneer replacement , flying from its big new carriers ?
We paid the penalty @ @ @ @ @ @ @ War .
The aircraft had a ferry range sufficient to get it to Ascension Island , from where it could have creamed Johnny Argie ’ s airfields , using inflight refuelling .
Not having TSR-2 meant losing lives .
How much better it would have been , rather than throwing away the lives of our servicemen in the entirely predictable Falklands War , when we really needed TSR-2 , to have executed Roy Jenkins and Harold Wilson in World War II , when both were working hard for a Nazi victory ?
Wilson @ @ @ @ @ @ @ As a serving officer in the Armed Forces of the King , Jenkins was entitled to the courtesy of being shot . )
Wrong though its conclusions may be , with respect , TSR-2 would make a fine addition to any library and has been added to mine .
Hugely entertaining and a real TV feast , Quiz was broadcast on ITV last week on three successive nights .
Starring Matthew Macfadyen and Michael Sheen it tells the story of the ‘ Coughing Major ’ , Charles Ingram , who tried to cheat @ @ @ @ @ @ @ Who Wants To Be A Millionaire in 2002 .
Ingram was indeed a major , in the British Army , albeit not from one of the better regiments .
Key events were changed for dramatic reasons , but the mini-series is essentially true to life .
The show has of course sparked debate as to whether or not Major Ingram ( he resigned his commission but was not cashiered ) and his wife are innocent .
Two clues about that don ’ t appear in the show – Ingram is a Remainer , @ @ @ @ @ @ @ to an unrelated insurance fraud in Bournemouth Crown Court .
Even though the trial was at Southwark ( it should have been held in Salisbury Crown Court , where as it happens I once prosecuted ) I incline to the view that the majority of the jury got it right .
( In most American jurisdictions the major and his wife would not have been convicted , by reason of a hung jury . )
I appeared in front of Judge Rivlin once ( as counsel , not as a defendant ! )
@ @ @ @ @ @ @ of the old school , and a just and considerate sentencer .
( The Ingrams received suspended sentences , by which I don ’ t mean hanging . )
However I think that ITV were wrong to call in the rozzers , who by and large couldn ’ t investigate their way out of a paper bag .
They were right to relieve Major Ingram of the cheque and cancel that night ’ s episode , but they should have left it there .
Prosecuting with an edited tape was always going to @ @ @ @ @ @ @ be referred back to the Court of Appeal .

Readers will not be taken aback to learn that I was surprised that PM Boris Johnson survived his brush with Covid-19. I knew that he was desperately ill and had been on a ventilator. I assumed, since he was in an NHS hospital, that he had been written off, along with all seriously ill NHS Covid-19 patients, and was being given palliative care only. It is now increasingly clear that he was treated with the anti-viral drug Remdesivir. If that is right the political consequences will be huge. Thousands of NHS patients have been denied effective treatment and have died. Once that gets out Boris is finished, no offense intended. The NHS won’t be far behind.
The General Medical Council were right to oppose the creation of the NHS. As they foresaw way back in 1948 socialised medicine would lead, inevitably, to decisions being taken by bureaucrats rather than doctors, in the interests of the state rather than the patient. In practice key decisions on NHS strategy in the Covid-19 outbreak have been taken by the Cabinet Office, which controls every aspect of British bureaucracy.
Sending the PM to an NHS hospital looked like a death sentence. On Cabinet Office orders the only drugs known to be effective in combatting Covid-19, Hydroxychloriquine and Remdesivir, have been withheld from NHS patients. At the same time government (in reality, Cabinet Office) policy, to which I am strongly opposed, is to sacrifice frontline NHS staff by withholding information that the virus is airborne. They’ve been going down like ninepins.
It didn’t occur to me that the Cabinet Office would sanction Remdesivir for the PM only. No offense to the PM, but far more important people, such as the actor Tim Brooke-Taylor, have been allowed to die without receiving proper treatment. Prime Ministers are important of course, but even the PM’s strongest supporters would scarcely suggest that he was worth a Goodie! (The Goodies was an hilarious 1970s comedy show.) Boris can be replaced. Tim Brooke-Taylor can’t.
The official British government line is that neither Hydroxychloriquine and Remdesivir have been proved to work in clinical trials. This is nonsense. The Marseilles trial I’ve been writing about was peer-reviewed and published in a reputable journal. It may have been small-scale but it was conducted properly. The drugs have been used to good effect in the US, China and now Britain. There is no need to hold up their deployment any longer.
It’s perfectly clear that Hydroxychloriquine is safer than Chloriquine and that even Hydroxychloriquine is not suitable for all patients, for example those with heart defects. It’s equally clear that it’s suitable for the majority of Covid-19 patients, particularly in the early stages of hospital admission. Remdesivir seems to be more suitable for patients in acute respiratory distress. Bureaucrats holding up the prescription of both drugs are playing with lives.
When was the President told?
We know that Covid-19 is a bioweapon, that it was released, unintentionally, from the Wuhan Institute of Virology in mid-November and that Peking covered it up for weeks, whilst tens of thousands of their people died. We also know that WHO covered it up as well. It’s also clear that the weapon was mainly designed to cause economic damage, not deaths.
CIA, who as revealed on this site were involved in the Obama-era Covid-19 program, working with the ChiComs, clearly knew in November. Equally clearly the White House were not told. It seems that the President wasn’t told until around the middle of January, around about the same time Covid-19 started to take off in the States. It was also the time of maximum distraction over that Ukraine nonsense.
Although the President is being strongly criticised by the Democrats for delay, given the circumstances he moved quite quickly. Insanely, the British government still hasn’t banned flights from China!
The mere fact the CIA knew that something was up in Wuhan most definitely does not mean that the White House knew. The Agency are strongly backing Joe Biden in November and were in the Russia and Ukraine hoaxes up to their necks. Almost certainly Langley thought that Covid-19 would be an ideal way to embarrass the President and increase Joe Biden’s chances. The fact that tens of thousands of Americans might die in the process doesn’t seem to have troubled them. The CIA are well used to writing off American lives.
It is not unreasonable to suppose that senior Democrats were briefed in by the CIA and were prepared to go along with the plan to keep the President in the dark. Are the Democrats that cynical? You bet.
This is the party that knowingly installed a non-citizen in the White House and then covered it up. It’s also the party that went along with an absurd conspiracy theory that Russia somehow hacked into the party’s server, when the download speed ruled hacking out completely, and when that nonsense fell apart they invented another hoax concerning the Ukraine.
It’s also the party which cynically relies upon African-American voters not being willing to go elsewhere and plans to throw them under a bus by giving their jobs to Hispanic immigrants. As that nice man Ted Hayes, an African-American activist in LA, explained to me when taking me around Skid Row in LA some years ago, it’s African-Americans who mostly suffer from illegal Hispanic immigration. Middle-class whites tend to benefit if anything, as they get cheap domestic labor. And, yes, I’ve been to Skid Row, where as a white upper middle class English barrister I was received with great civility and good humor. You tend to meet nicer people there than in Hollywood.
Of course the Democrats are prepared to throw working-class whites under a bus as well, by taking their votes in the rust-belt states and then exporting their jobs to China. They’re so desperate to win in November that they’re even prepared to indulge in large-scale voter fraud. (Then they scream blue murder when the Administration takes sensible measures to prevent it.)
I have little doubt that senior figures in the Democratic Party were alerted to the Wuhan outbreak by the CIA as far back as November and planned to take political advantage of it. I suspect they also knew that the Agency, CDC and DNI had all agreed to hold this vital intelligence back from the President. CDC have about a dozen people embedded in the evil, genocidal World Health Organisation, no offense intended, and were in the loop by December at the latest.
WHO
President Trump was absolutely right, with respect, to withhold funding from WHO. In so doing the President has shown real leadership. Other Western countries should follow America’s example. It’s high time that the UN and its agencies were given a real smack.
WHO have not only covered up China’s role in creating Covid-19 (with CIA assistance of course), but her role in creating SARS and MERS as well. Working with CDC in Atlanta they have also covered up Germany’s role in creating the Spanish Flu bio-weapon.
WHO’s shameful actions have cost many lives. Bio-weapons typically are airborne, because they are genetically engineered for easy transmission. Hiding their origin not only protects those states which develop them, but puts medical personnel at risk. The final death toll from WHO’s actions is likely to be in the tens of thousands.
The Beatles and the Supreme Court
The Beatles are back in the news. Not the long-haired lovers from Liverpool but the murderous ISIS terrorists who liked to chop people’s heads off. Maha Elgizouli, the mother of one of these clowns, has outrageously secured a with respect ludicrous decision from the UK Supreme Court effectively preventing British witnesses from giving evidence at the trial of her son, Shafee El Sheikh. (Elgizouli v. Secretary of State for the Home Department [2020] UKSC 10)
It has to be said that there’s nothing much ‘alleged’ about El Sheikh’s role as an ISIS executioner. His supporters aren’t seriously arguing that it’s a case of mistaken identity. Their argument is that the Sudanese terrorist should be given a soft sentence, presumably with a view to encouraging other young Islamists to go around chopping people’s heads off.
As a result of this decision it’s now unlikely that you guys will be able to prosecute. The terrorist-sympathising Crown Prosecution Service, no offense, has already decided that he cannot be prosecuted in the UK. Since he’s no longer a British Citizen he has no right of entry into the UK in any event. He won’t be coming back here. It looks like he’ll be headed to Gitmo.
One option of course would be to hand him back to the Syrian Democratic Forces. They would have jurisdiction, since they captured him on the battlefield. They could give him a fair summary trial by court martial and shoot him, nicely of course. (No need to chop his head off.) Since the Beatles believed in videoing their executions there’s no particular reason why the SDF couldn’t broadcast the execution on YouTube, subject of course to any rules YouTube may have about broadcasting live executions.
Another option would be to amend the UK Data Protection Act and make it clear that personal data can be transmitted to states with which Her Majesty enjoys diplomatic relations for law enforcement purposes regardless of that state’s sentencing policies. This would get around the Supreme Court’s finding that the Data Protection Act was somehow breached by sending witness statements and other material to the Justice Department.
The reasoning, if reasoning is not too strong a word with respect, of the justices is unlikely to stand the test of time. Their interpretation of the Data Protection Act is strained to say the least. Lord Kerr in particular strongly disagreed with the softening of the British government’s opposition to the death penalty, banging on about the policy of previous governments as though it mattered. Lord Kerr, with respect, may have preferred the policy of the Blair and Brown governments, but they’re out of office.
Lord Kerr, a half-crazed Euronutter, no offense intended, also made the hugely controversial comment, likely to be the court’s death-knell, that now that we’re out of the EU the judges can bring back community law by the side-wind of declaring it to be part of the Common Law, against the will of the people and Parliament. With the greatest of respect Parliament should exercise its undoubted right to remove Lord Kerr from office and abolish the court. We should go back to having the House of Lords.
The House of Lords, with respect, was not immune from handing down silly decisions, Factortame and Metric Martyrs being cases in point, but none of them was this crazy. Basically what the Supreme Court has said is that it’s OK to go around chopping innocent people’s heads off. Maybe their perspective might change if one of the justices were to be captured by ISIS.
This week’s reading: TSR-2: Britain’s Lost Cold War Strike
Aircraft (Tim McLelland, 2010, rev. ed. 2017)
Revised and updated, with an additional chapter on the F-111K by Tony Buttler, this is the most comprehensive work ever published on one of Britain’s greatest ever technological achievements, the superb British Aircraft Corporation TSR-2. Tim, sadly, died in 2015.
TSR-2 is a technical tour de force, giving a detailed description of the aircraft’s specification and advanced systems. There are excellent line drawings and fine studies of the prototype, XR219, flying from Boscombe Down and Wharton. Most of those flights were flown by English Electric’s brilliant chief test pilot, ‘Bee’ Beamont, a friend of a friend, who did superb work in ‘Tiffies’, strafing Jerry after D-Day.
Sadly however the book is let down by the failure to consider the intelligence aspects. Both the Air Minister, Roy ‘von’ Jenkins, and the Prime Minister, Harold ‘von’ Wilson, who essentially took the fateful decision to cancel the project between them, were German agents. The Defence Minister, Denis Healey (whom I once met at a Labour Party conference) unfairly took most of the blame. Denis was a nice chap, but he was a house-trained idiot, no offense intended, who swallowed the Treasury’s line that we couldn’t afford the aircraft.
As I argue in Spyhunter Whitehall never intended the TSR-2 to go into production, any more than they really intended to buy the F-111. The intent all along was to concentrate resources on one combat aircraft, cancel it, smash up the UK’s aerospace sector and transfer the technology to the Soviet Union. The plan worked, sadly.
‘Von’ Wilson’s talk of the “white heat of the scientific revolution” was just that – talk. Wilson wanted to deskill the UK and hold back our technological development. TSR-2’s replacement was a batch of old Blackburn Buccaneers transferred from the Navy after Labour’s equally disastrous decision to cancel the CVA01 and CVA02 attack carriers. Fine aircraft that it was, when first used, by the South Africans, to drop a live nuclear weapon, the aircraft was blown out of the sky by the shock-wave.
There is no doubt that a TSR-2 would have survived the delivery of that particular nuclear payload. It was capable of a staggering Mach 1.2 at 250 feet and could have egressed at Mach 2.2 at altitude having delivered a nuclear strike package. The aircraft not only met its hugely demanding specification, it exceeded it. It could take off from an unprepared strip no more than 3,000 long, fly more than 1,000 nautical miles, accurately deliver a nuclear or conventional payload with a high-lo-lo-high mission profile, with supersonic approach to the target, and fly 1,000 nautical miles back without inflight refuelling.
Its nav-attack and recon systems, including inertial navigation, line-scan camera and sidescan radar, were the most advanced of any aircraft in 1964. When flying asymmetric, on just one of her mighty Bristol Siddeley Olympus 22-R engines Bee managed to pull away from Jimmy Dell in the English Electric Lightning T4 chase plane. The finest interceptor in the world in the mid-60s, the Lightning was a Mach 2-capable aircraft with a rate of climb of 50,000 feet a minute. Arguably no other aircraft in existence could pull away from a Lightning on just 50% of combat power.
Staggeringly, the TSR-2 could also climb at 50,000 feet a minute, clean, with no external stores or tanks. That’s nearly a 1,000 feet a second, for a bomber. McClelland gives the lie to the claim that the TSR-2 lacked maneuverability. Its wing-loading was less than the F-111’s, which was nominally a fighter, hence the ‘F’ designation. At altitude a TSR-2 could out-turn most fighters of the era. It handled superbly.
Interestingly, McClelland reveals that the aircraft had provision for an arrester hook. Its heavy-duty undercarriage could have coped with carrier landings and it was stressed for catapult launch, indeed it was a very strong aircraft, designed as it was to fly supersonic close to the ground, using terrain-following radar. Were the Navy at one time thinking of TSR-2 as a Buccaneer replacement, flying from its big new carriers?
We paid the penalty for not having TSR-2 in the Falklands War. The aircraft had a ferry range sufficient to get it to Ascension Island, from where it could have creamed Johnny Argie’s airfields, using inflight refuelling. Not having TSR-2 meant losing lives.
How much better it would have been, rather than throwing away the lives of our servicemen in the entirely predictable Falklands War, when we really needed TSR-2, to have executed Roy Jenkins and Harold Wilson in World War II, when both were working hard for a Nazi victory? Wilson clearly should have been hanged. (As a serving officer in the Armed Forces of the King, Jenkins was entitled to the courtesy of being shot.)
Wrong though its conclusions may be, with respect, TSR-2 would make a fine addition to any library and has been added to mine.
This Week’s TV Review: Quiz, ITV, Airdate April 13th 2020
Hugely entertaining and a real TV feast, Quiz was broadcast on ITV last week on three successive nights. Starring Matthew Macfadyen and Michael Sheen it tells the story of the ‘Coughing Major’, Charles Ingram, who tried to cheat his way to a million quid on Who Wants To Be A Millionaire in 2002.
Ingram was indeed a major, in the British Army, albeit not from one of the better regiments. Key events were changed for dramatic reasons, but the mini-series is essentially true to life.
The show has of course sparked debate as to whether or not Major Ingram (he resigned his commission but was not cashiered) and his wife are innocent. Two clues about that don’t appear in the show – Ingram is a Remainer, no offense intended, and pleaded guilty to an unrelated insurance fraud in Bournemouth Crown Court.
Even though the trial was at Southwark (it should have been held in Salisbury Crown Court, where as it happens I once prosecuted) I incline to the view that the majority of the jury got it right. (In most American jurisdictions the major and his wife would not have been convicted, by reason of a hung jury.) I appeared in front of Judge Rivlin once (as counsel, not as a defendant!) He was a very fair tribunal, of the old school, and a just and considerate sentencer. (The Ingrams received suspended sentences, by which I don’t mean hanging.)
However I think that ITV were wrong to call in the rozzers, who by and large couldn’t investigate their way out of a paper bag. They were right to relieve Major Ingram of the cheque and cancel that night’s episode, but they should have left it there. Prosecuting with an edited tape was always going to be problematic and the case may yet be referred back to the Court of Appeal.


veteranstoday--2020-04-18--Documentary Proof:  University of North Carolina Generated COVID-19

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 @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ , 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 ) .
Similar 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 ) .
Examination @ @ @ @ @ @ @ 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 ) .
@ @ @ @ @ @ @ , 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 @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ ( 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 .
@ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ 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 with regards @ @ @ @ @ @ @ 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 to vaccination @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ .
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 @ @ @ @ @ @ @ 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 to mediate @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ 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 incorporated changes @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ 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 that are @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ 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 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 @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ 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 restrictions @ @ @ @ @ @ @ 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 @ @ @ @ @ @ @ 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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