Saturday, February 29, 2020

Meanwhile At Costco, The Hoarding Begins

The switch is starting to move. Seems that Sam's Clubs, Costcos, and Targets are being emptied as of TODAY, leap year day, 2020. By week's end, every food and clothing and hardware outlet will have its shelves stripped of food to gas cans. The average time clean out a Costco in NY? Two hours, then empty.

The crowds started showing up a half an hour before the places opened.

Sam's Club and Costco to panic buy food and health supplies as virus fears surge.

DB

------------------------

The same long lines that we've seen in China, Japan, South Korea, and across the world as people panic buy food and health supplies have started in the US.
On Saturday, the US Surgeon General urged people to "stop buying masks," saying on Twitter that they're not effective in preventing the general public from catching coronavirus.



Despite the CDC telling everyone to calm down, alleged video of long lines pouring out of a Costco store in Brooklyn, New York, surfaced on YouTube Saturday afternoon.
https://www.bitchute.com/video/NMyLKGJLpM9B/
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Event 201 Pandemic Exercise: Segment 2, Trade and Travel Discussion
2nd hour highlight notes.

Significant escalation.
  • 13,000 deaths
  • Massive underreporting.
  • Projection in month 2million cases, 100,000 deaths.
  • 3 month projection 20 million cases, 1.5 million dead.
  • Markets in the Red.
  • Havoc in Trade and Travel.
  • Sick are overwhelming healthcare facilities worldwide.
  • No people in public out of fear and authority reccomendations.
  • Retal/service sectors fucked.
  • Businessess struggle to operate. They're fucked bigly.
  • Basic services are fucked as workers sick.
  • Travel sector/tourism fucked. 'Some' countries banned travel from worst hit countries.
  • Manufacturing hit as people not buying shit they don't need.
  • Food, medicine being hoarded by public.
  • Economy fucked. Stock Markets fucked.
  • 30 x Sars cases and 100 x Mers cases.
  • Human and trade travel bans not stopping spread.
  • Some countries banning goods from heavily infected countries. Supply chain distruptions.
  • Global GDF to drop 4 percent - Global recession inbound.
  • High unemployment and runaway inflation would result.
  • Stopping trade/travel means grave economic consequences.
  • People will travel anyway.
  • "This is only going to get worse"
  • Supply chain workers refusing to work unless masks, protection.
  • Use social media to 'calm' people/public.
  • Reporters can't get into infected areas to report accurate info.
  • Travel/trade disruptions means cannot bed precursor medication to make drugs for every other non-virus medications (schizo meds)
  • WHO suggesting NOT to implement travel bans. (WTF?)
  • Supply chain disruptions - countries nost accepting ships etc with goods from harder hit countries.
  • Airlines cannot maintain flying with low numbers of passengers. Flights cut.
  • Suggesting governments should bailout, give Airlines, travel affected entities with financial help.
  • Once again suggesting U.N/WHO should be collective management.
  • U.N. to 'help' implement accurate reporting in affected countries.

Relax...we wouldn't lie to you. Sincerely, China



STOP WORRYING. STOP WORRYING.
STOP WORRYING.
STOP WORRYING.
STOP WORRYING.
STOP WORRYING.
https://www.bitchute.com/video/rVeMaiwntVVC/

Herd immunity:
 

To combat wildfire, you have two options: throw water or prevent it from spreading by digging trenches, chopping wood down, etc. herd immunity is the same thing. When enough people are immune to a certain disease, that disease is unable to find hosts to infect and thus, cannot spread. There's no herd immunity for Corona-Chan.

When the weapon part of covid hits

https://www.bitchute.com/video/Rr8zY0gfgB1X/ 

Your government however has neither the means to deal with this in any effective way or particular inclination to stop the culling of 50-100 million extra baggage in the population.
It would be impossible especially currently to try china-style lock-downs using armed forces in India, Pakistan, or Bangladesh. It is even more impossible to somehow ensure any within such zones can get adequate care to increase their chances of survival. 


Only those who have prepared and stay away from virus breeders have a chance. A slim chance, but it's at least there. But then there are those who are prepared, but are too Fing lazy to spray down door handles on their houses, cars, shoes. et al. They wear clothes and shoes that cannot be sprayed, because they "look cute" and are suade. These people are the real danger in any prepared situation. Their laziness will kill the whole house. And there is always one, who doesn't use the ozone, who doesn't spray down surfaces, and doesn't wash clothes and change...because THEY ARE FUCKING INHERENTLY LAZY SHITS and their selfishness will kill everyone around them. Give them the facts of life or show them the door, before their laziness kills all your efforts toward survival.

Your entire urban and populated zones are kill zones for the virus, with government having little means to keep the public in line or cooperative. In fact, by the obvious "not here" attitude in the west, they damn well know millions will die and THEY WANT THAT.


Therefore, best they can do is pretend nothing is wrong, hoping people will pass it off as 'another dead body because etc'.


All this is based on the idea that short term (6 months) they can keep this hush hush until the cures and shit comes out. If it doesn't, only then will your government take overt action.
Hell they are barely testing people because not enough resources in India. Right now, India should be were italy, SK is regionally but no one is really paying attention or caring because they don't give a shit about each other.


Being a leftist PC is suicidal and murderous

DB 02-29-20 Leap year



Out smashing satanic altars, about a dozen we found in the hillsides that day. Doing my part to piss off wiccans and witches, everywhere. I am always amazed at how many we find, doing this work.
Ignore that man in the picture above, trust your government

https://www.bitchute.com/video/rVeMaiwntVVC/

Good morning all,

What can I say? There more I dig, the uglier it gets. This information brought forward is for all our awareness so we may each of us, act accordingly in protecting friends and family, from the plague. To call it anything else is to undermine the seriousness of this world event. 
67,368,000 bodies per week. Hubei and Wuhan, see math below in related section.

It's the sabbath today. My heart is full of gratitude, Yeshua, and a nice smile at all the folks out there who report in on what is going on in their country, locale, whatever. This anecdotal, honest seeing of things AS THEY REALLY ARE, helps all of us in facing this crisis. And preparing so that we may help and serve others, who are constantly lied to by the media, the governments, and by the NWO Illuminati Network. Damn them.

Also, many of you are doing everything you can, taking risks, and doing whatever...for others. Dad's blessing be upon you all, I pray our Heavenly Father. You are the best. In the worst of times. In point of fact, many are helping me, help others. With food purchases, funds so that others can have protection, and of course, putting themselves in harm's way. These are great souls in the making, tried in the furnace of life and death, with pure gold and silver, the outcome. As Dad said he would do to this last generation. I am very grateful and glad to know you all. You are the smile on my face, all of you.

Let's get to it. Sorry, no upsides today. Fair warning, what follows isn't pretty. But in the knowing, you find freedom with truth. So when the paid liars come at you, their arrows do not land. I beg you, free others with your knowing. Please.

Longtime reader in Brazil to start today off, sent this to me in the wee hours.


  • "Last Tuesday Brazil announced his first confirmed case, the "virus" is the same from Italy. Timing was suspicious as it happened right after Brazilian carnival. So I'm keeping watch to Italians to see if the mortality and chaos are similar to China. Since the 1st case, it took Italy about 20 days, 2 deaths and 80 confirmed cases until real city lock-downs happened. Off course this are the official numbers. So that might be the time window before they start implementing more "draconian" measures here. I might get trapped in a different city than my family because of this. So far they are telling the "just a flu don't worry" story, so population is not very worried yet. But an online run to buy a mask has already happened and it's getting hard to get an n95. I've got mine. So there is some mistrust about the government.

  • "The first Brazilian case was identified in the Israeli Albert Einstein hospital in the city of São Paulo (in the same state where I live). This is the same place which operated our president's (Captain Bozo) bowel cancer, under the guise of a false flag knife attack, on September 7th 2018, independence day in Brazil. So it's likely the virus case is a bogus one to justify the use of their bio-weapons in Brazil.
  •  "In China, the mandatory vaccinations law took effect in December 1st. The oldest confirmed case was backtracked to December 1st, although some think the outbreak was probably already going since October. (we already know. DB)
  • "In Brazil, today government announced an anticipation of 23 days of the flu vaccination campaign, which will start on March 23th. 23 and 23, this is no coincidence.They intend to use the flu shots to help medics discern common flu from coronavirus cases during diagnosis. So you get the flu shot and you show up sick and they know for sure YOU HAVE CORONAVIRUS and not the flu!!! Bull shot! That's their plan, spoken today by the minister of health himself in a press conference. So we might expect deaths to really start ramping up after March 23th. And the main target of the campaign are elderly people, which is the announced group risk for the virus disease. All fits perfectly. Very convenient for a country that just supposedly had a retirement fund crisis to just get rid of elderly people.
  • "In the meantime I see BBC news of China under reporting the cases and how horrible Chinese quarantines are. Why are they telling this? Are they trying to scare westerners? Is the west version of the weapon different enough for this to be necessary? Jimstone freelancer is reporting about a binary weapon that has pork as one of the components and triggers lung inflammation with sticky clear phlegm and no fever. He writes he saw evidence of it being used only in China so far. If that is so, there might be hope for the common people after all elsewhere in the world. Hard to confirm this though.

So Brazil is about to get a flu vaccine. Like China did, and the date? Witches sabbath, first day of spring, Passover, all in the zone. They love their satanic timings of things. I guess the death numbers in China, which I will cover shortly, are such a success for these satanic killers, they are doing it everywhere now. I fully expect mandatory flu vaccines in at least California, Washington, NYC, big metros and states. 

They will, in the US, make it a mandatory on this wise.
  1. get the shot. If not, then
  2. No buying or selling,
  3. No public travel,
  4. health inspections of your domicile, where they then decide
  5. if you not taking the shot, is endangering the lives of others. Then they take you away anyways, in that van with no windows. DO NOT GET IN THE VAN. You will never be seen again alive.
This will be the gun they hold to our collective heads. It's what they did in China.
  • Hospitals in Seattle, Los Angeles, San Diego, etc are all reporting plague victims. It's just the numbers that are the lie. They always say the same thing - unknown origin. Sure. They know. As much as they are trying to hide the plague here, it's now at a point where the numbers are getting too big to keep things buried. We are very nearly to that day of the light switch, when it all changes overnight. Store runs, empty streets, hospitals with lines going a mile down the road. That's how it started in China, when there light switch day happened. That's the first week, even the second week.
  • Oregon Officials Confirm Third Coronavirus Case "Of Unknown Origin"; Risk Of "Community Outbreak" Is High.
  •  I am convinced at least 15% of the population I see around me already has the virus. And now, as incubation times come and go, sure enough they are showing up at their doctors and hospitals, sick as dogs. It's already happening in California.
  • Health authorities in Texas and Oregon report 12 new coronavirus case. Only 12? Like China, America intends to lie about the numbers, while it starts burying people by the truckload.
  • US coronavirus case total hits 63, 2nd case 'of unknown origin' confirmed. These low numbers are just offensive to me. Offensive.
  • WHO says 20 vaccines in development. You mean you want to slam this poison in the veins of billions of people and kill as many as possible, because that's what the WHO has done over the years in Africa. Go to villages, inoculate the locals, they get sick, and a third of them die. Go to next village.









  • "This Is A Nightmare": London's Worst-Case Plan For Coronavirus Includes Giant Morgue In Hyde Park
  •   
  •  

    It's Time To Stop Pretending That In All Of Africa There's Only 3 Covid-19 Cases

     

    Update: Fears that the coronavirus outbreak will be officially declared a global pandemic have grown after the first case was confirmed in sub-Saharan Africa. Nigeria’s health ministry said the case was found in Lagos, one of Africa’s biggest cities, with a population of 20 million people.

    Africa has recorded only two cases up to now, one in Algeria and one in Egypt.
    The spread of contagion to countries with poor public health systems has alarmed the World Health Organization, which said it could “get out of control”.


















    *  *  *
    Via Golem XIV's blog,
    Surely it is time to stop pretending that in all of Africa there are only three cases of Coronavirus (1 in each of Algeria, Egypt, and Nigeria)?  Everything we now know about the virus makes this virtually impossible to believe.

    We know that despite alarm being expressed by other African nations, Ethiopia has continued to keep open its air link to China via Bole International airport. The Ethiopian authorities have justified their decision by quoting the WHO president’s ‘expert advice’ that travel bans were not going to stop the virus and therefore were not recommended. As reported by Global Research,
    … he made the inexplicable statement that other countries were not warranted to ban air travel to China as precaution. He declared,” It’s not a time for judgment… This is a time for solidarity, not stigma,” refusing to recommend any international restrictions on travel or trade with China.
    This seems very odd and ill-advised ‘expert advice’ when at the same time the official line is that while we may not be able to stop the spread of the virus we should strive to slow it down to give us as much time as possible to look for treatments and a vaccine.
    What else do we know? Well we can see from recent events in Italy that the virus spreads very fast before anyone is likely to show any symptoms. We also know how easy it is for over-worked doctors to not pay enough attention to symptoms which on their own  are not all that serious to begin with and could be the result of the common cold or ordinary strains of flu.  The Corona virus does not present with the kind of dramatic symptoms that make it easy to spot.
    We also know that the two things about this virus which makes it very dangerous and almost impossible to stop. The first is the very  long, unusually long, suspiciously long latency period when someone can carry the virus and be highly infectious without showing any symptoms at all. The second is that Covid19 is easily transmitted and very highly infectious.
    Despite having all the advantages of wealth and warning none of the best prepared, best off countries have been able to stop its spread. Is it believable that the Coronavirus is not already spreading in African countries?
    If Coronavirus is spreading in Africa then everyday we hear no reports of it, is another day it is spreading unreported and untreated. Is this alarmist?  I certainly hope so but common sense tells me its just tragically very likely.
    The latency period before someone shows symptoms means that it is quite possible for someone to be carrying the virus but show no signs whatever when they arrive at the airport and are screened. If they did have the virus then given its infectivity it is highly probable that other passengers being in close contact in a sealed environment like a jet for many hours are going to be infected. None of them will show any symptoms when screened. How many of them will travel on to other airports, other cities other towns where there are fewer if any facilities for testing?
    Could this scenario happen? Well  we already have the case of a Chinese worker to flew into Kenya, who was not stopped or quarantined at the airport and travelled on to a road construction site in Kitui county outside Nairobi. Why was he not stopped? Well for the perfectly good reason that he was not showing any symptoms.  The case came to light because when the man arrived at the construction site,
    …road workers contracted by Sinohydro Corporation reported a case of a visibly sick Chinese national in their construction camp.
    The Kenyan article went on to say that his colleagues were told to isolate him and observe him but not to come into contact with him.
    The case made the papers in Kenya because when local police arrived at the camp they were denied admission by the Chinese company Sinohydro Corporation. It was also reported in the Kenyan press that a local medical team were also denied access.
    Medics denied entry to Sinohydro Mutomo camp
    Kitui County Chief Officer for Health Richard Muthoka confirmed that his medical team was also denied entry to the camp.
    “Our medical team wasn’t allowed to access the Chinese camp in Mutomo but we have taken the necessary measures to swiftly establish what is happening and ensure all people working and mingling with the foreigners are safe,” Dr Muthoka stated.
    Its interesting and important to note that an article in the ChinaDaily refuted the story the very same day. According to the Chinese article the man was not sick and his quarantine was routine. Which may well be true.
    All this was 10 days ago. There has been no update that I can find. But whether the man was carrying the virus and was sick or whether it was all routine precaution is not really the point. Given the long latency of the virus we know from what has happened in Italy that the man could have been carrying and spreading the virus. Even if he was not how likely is it that no one entering Africa has been?
    If I am in any way right, and I fervently hope I am not right, then all the headlines telling us how China getting its epidemic under control will not really matter from a global point of view. Of course it will be a huge triumph for China and I hope they succeed. But because the world seems to be turning a  blind eye to what might be going on unreported in Africa,  we could soon find that we have merely substituted one huge reservoir of infection for another. And Africa, unlike China is not going to be able to contain it.
    In South Africa which is considered one of the African countries better equipped to deal with the virus this was the headline in The South African.
    Coronavirus in Africa: Scare in Namibia, as SA hospital ‘not ready’ for cases
    Tembisa hospital has been designated as one of the facilities where Cornoa virus victims would be quarantined and treated. The only problem is as the paper reports,
     The 44-bed neo-natal unit is often overcrowded, and staff have previously complained about their working conditions, casting doubt over Tembisa’s ability to host coronavirus patients.
    The report goes on,
    Jack Bloom, the DA’s leader in Gauteng, is upset that this location was chosen as one of three Gauteng hospitals to house patients with the deadly disease:
    “Tembisa Hospital has been identified as one of three hospitals in Gauteng that will treat coronavirus cases, but this hospital is severely overcrowded and is only just recovering from antibiotic-resistant klebsiella infections that caused the deaths of 10 babies.”
    “Unlike the other two identified hospitals, Charlotte Maxeke Johannesburg and Steve Biko, there is simply no space at Tembisa Hospital to isolate coronavirus patients. It cannot be that Tembisa Hospital was chosen simply because it is near the OR Tambo airport where coronavirus-infected patients could enter the country.”
    Meanwhile in Rwanda, 

    Rwanda health minister fired

    Rwanda’s president Paul Kagame on Sunday … who was speaking at the National Leaders’ Retreat…accused several of them [ministers] including the ex-health minister Diane Gashumba of lying… to him about the availability of kits to test individuals for the deadly Coronavirus, Covid-19.
    “One morning, I called one of the leaders and the Prime Minister about the Coronavirus and asked that they examine each one of us ahead of the Leader’s Retreat. I told them to tell the minister of health to ensure this."
    “She responded that we have 3,500 kits, and that using 400 of them to test leaders would seriously deplete the number of kits we have. …
    “Later we discovered that we do not have the kits as she said. We have kits for only 95 people and not 3,500.
    If the world stands by and allows the virus to spread in Africa then Africa will be the reservoir for the virus from then on.  We will have a situation where every traveller, every migrant and refugee trying to enter Europe from Africa, arriving on beaches in Spain, Italy and Greece, will likely also be a carrier of the Corona virus. That is a situation that will turn very ugly very fast.
    Ironic and horrible as it is to say, the only saving grace might be that the virus will have already become endemic in Europe before that happens.
    So much for Africa and Europe. What of America? There too the virus is going to reveal ugly things that people don’t want to face up to.  It is just a fact that America has third world levels of poverty and neglect in almost every city. What will happen when the virus gets into those forgotten populations?  Will very crack head and meth tweeker self isolate for the public good? Or will they be driven by hunger and a desperate need for their fix to be out every day, sick or not, many turning tricks and all looking for a fix?  How is that going to work?
    Will America lavish upon those people a level of medical care that they have never given them before?  Suddenly the people living in tents and under freeways, defecating on city streets because they have nowhere else, are they going to be given hospital beds and isolation units?
    Lets suppose the FEMA camps are opened up. How is it going to work putting those people, the people we prefer to forget, in the same camp as your grandmother?  Social cohesion?
    This virus is acting like an x-ray in all our countries and what its showing is not pretty.
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The screaming at night in Hubei and Wuhan. https://www.bitchute.com/video/eueD5Z99MUDq/ Goes for hundreds of miles in every direction, it is reported. These are the survivors, the people who have watched and are watching, others die. And for two months now. And, all they have left is their horror. Many are throwing themselves out the window, to their deaths, rather than go through another day of this endless, heartless, horror, China is putting them through


    Because we know, they don't get water, or power, or food, if they test positive. This means that once you get the thing, it's a foregone conclusion that the government has already written you off and is just waiting for the "community" managers to come get your stiff. This is so, because they are so overwhelmed with the dead and dying, they just leave you alone to die in your welded shut apartment. These people know they are doomed and there is no way out of it, though some try. Those that try, get picked up and executed by the CCP.
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The latest daily numbers have grown so exponentially in the last 2 weeks that it boggles the mind. Here they are

  • 400,000 new incinerators already in place, 1,000,000 more being built and to be deployed in the next week alone, nationwide.
  • 912 existing funeral homes and crematoriums. This includes hospitals. Hubei and Wuhan numbers only. Remember this. This is just for that area, which is ENORMOUS.
  • Each unit processes 1 body per hour 
  • x 24 hours a day
  • That's about 401000 x 24 equals
  • 9,624,000 bodies every 24 hours, or per day.
  • x7 days equals
  •  67,368,000 bodies per week.
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 70% of 600 contacts without symptoms test positive
http://archive.is/Fg3tL

Corona infects China's Great Fire Wall
http://archive.is/V3P0B

Indian man tests negative, dies of pneumonia next day
http://archive.is/HpyTu

2100 dead in Iran, regime covers it up
http://archive.is/D7YcK


 Asymptomatic concentrate virions at nose, more infectious
http://archive.is/RypEi

HIV-like mutation made it 1000 times more infectious than SARS
http://archive.is/xwQVe

SARS-CoV mechanism for antibody-dependent enhancement
http://doi.org/10.1128/JVI.02015-19



China figures likely simulated
http://archive.is/gzUVo

WHO: Detailed information provided in only 38% of cases
http://archive.is/GSeHM

nCoV "suspected" deaths outnumber "confirmed" 6 to 1
nhttp://archive.is/Lb6ry
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This virus is like a bio-weapons researcher's dream come true.

1.  Infectious for weeks before the patient shows symptoms
2.  Highly contagious.  It appears to be several times as contagious as the flu
3.  Will survive on smooth surfaces like doorknobs, faucets, phones for 9 days
4.  Survivors don't have immunity.  14% of the "recovered" had already caught it again when they went for follow-up checkup
5.  It is cross-species compatible; mammals, reptiles and birds are all capable of getting infected.  It supposedly originally came from bats, but that is one of the few species that the Wuhan fish market doesn't sell.  Among the possible "intermediate" species that Chinese authorities suspect are snakes, lizards, turtles, marmoset, rodents.  And we now know that, when they ordered pet owners to turn in their pets for disposal, it was for good reason:  Dogs in Japan and Hong Kong have tested positive.
6.  Even if you recover, you will probably have lung (81%), heart (38%) or kidney (28%) damage
7.  Outside of China, there is a 16% death rate among people who have an outcome.  China keeps their death rate at 2% by dumping all of the people who are still sick in the same pile as "recovered".  That's like if a hospital for the terminally ill claimed that they have a 100% successful recovery rate because none of the dying patients had actually kicked the bucket yet.  Also, an abnormally large number of the virus patients in Wuhan have died of heart attacks, kidney failure, or lung disease instead of the virus, so they end up in the "recovered" pile....
This is a virus that will kill about 16% of the people it infects, leave survivors with permanent damage and no immunity, is easier to catch than the common cold, is infectious for weeks before victims show symptoms, can survive on commonly touched surfaces for over a week, can be transmitted by pets, bird poop and mice, and binds to human cells 1,000 times easier than SARS.  Even in their wildest fantasies, bio-weapons researchers couldn't have dreamed up a better weapon...
The core of this virus is identical to a "chimera" coronavirus that is capable of incorporating snippets of various other viruses like SARS, MERS, HIV, H1N1 that was created at University of North Carolina, funded by a grant from the Wuhan level 4 infectious disease lab, which also sent two of its researchers as consultants.
-------------------

Corona-fear: France bans all mass public gatherings in confined spaces as German, Swiss fairs canceled 

 https://www.rt.com/news/482017-france-bans-mass-gatherings-coronavirus/

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BREAKING: LOMBARDY REGION ASKING RETIRED DOCTORS TO WORK IN RED ZONE HOSPITALS, doctors by the hundreds, coming down ill with Corona plague in Italy.
RIP ITALIANS


When looking at the genome sequence of the new coronavirus, Professor Ruan Jishou and his team at Nankai University in Tianjin found a section of mutated genes that did not exist in Sars, but were similar to those found in HIV and Ebola.
>In a follow-up study, a research team led by Professor Li Hua from Huazhong University of Science and Technology in Wuhan, Hubei province, confirmed Ruan’s findings.
>The mutation could not be found in Sars, Mers or Bat-CoVRaTG13, a bat coronavirus that was considered the original source of the new coronavirus with 96 per cent similarity in genes, it said.
>This could be “the reason why SARS-CoV-2 is more infectious than other coronaviruses”, Li wrote in a paper released on Chinarxiv on Sunday.
>Meanwhile, a study by French scientist Etienne Decroly at Aix-Marseille University, which was published in the scientific journal Antiviral Research on February 10, also found a “furin-like cleavage site” that is absent in similar coronaviruses.


From Italia Daily telegram:
Okay this is very big.
Basically Lombardy Region is looking with the central government if it's necessary to recall retired sanitary personnel in order to ease the work of the already stressed staff.
Some medics are working even 30 hours shifts.
This will have various results:
- Current medical personnel in non-used hospitals may be called up to take the turns of the overloaded personnel.
Some hospitals have been defined as "non-usable" in the fight against the virus, as those will be needed to perform the various operations of the used up hospitals.
Things like surgeries and medical checks are provided by those hospitals.
- Called-up-retired-personnel very probably will be used to replacement of current staff in "non-usable" hospitals.
- Students in the medical univestities may be called up as "fresh meat" in hospitals soon.
Probably the best students will be sent into the "usable" hospitals, while the others will be used in the "non-usable" ones.
> System overload soon.


--------------- 



Friday, February 28, 2020

China is building 1 million incinerators

Coronavirus - Patented Weapon

Uh oh, looks like he picked door number 2, the one with the bio weapons.
See you later. Now we see if they actually have an antidote or does the guy experience like the rest of us. Cuz he's really high up in the network food chain so we'll see.

Corona Pope
https://www.bitchute.com/video/P9kRvNaAhOVY/ 

The present invention provides a live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or more of non-structural protein(s) (nsp)-10, nsp-14, nsp-15 or nsp-16. The coronavirus may be used as a vaccine for treating and/or preventing a disease, such as infectious bronchitis, in a subject.



Description
FIELD OF THE INVENTION
The present invention relates to an attenuated coronavirus comprising a variant replicase gene, which causes the virus to have reduced pathogenicity. The present invention also relates to the use of such a coronavirus in a vaccine to prevent and/or treat a disease.
BACKGROUND TO THE INVENTION
Avian infectious bronchitis virus (IBV), the aetiological agent of infectious bronchitis (IB), is a highly infectious and contagious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct. IBV is a member of the Order Nidovirales, Family Coronaviridae, Subfamily Corona virinae and Genus Gammacoronavirus; genetically very similar coronaviruses cause disease in turkeys, guinea fowl and pheasants.

Clinical signs of IB include sneezing, tracheal rales, nasal discharge and wheezing. Meat-type birds have reduced weight gain, whilst egg-laying birds lay fewer eggs and produce poor quality eggs. The respiratory infection predisposes chickens to secondary bacterial infections which can be fatal in chicks. The virus can also cause permanent damage to the oviduct, especially in chicks, leading to reduced egg production and quality; and kidney, sometimes leading to kidney disease which can be fatal.

IBV has been reported to be responsible for more economic loss to the poultry industry than any other infectious disease. Although live attenuated vaccines and inactivated vaccines are universally used in the control of IBV, the protection gained by use of vaccination can be lost either due to vaccine breakdown or the introduction of a new IBV serotype that is not related to the vaccine used, posing a risk to the poultry industry.

Further, there is a need in the industry to develop vaccines which are suitable for use in ovo, in order to improve the efficiency and cost-effectiveness of vaccination programmes. A major challenge associated with in ovo vaccination is that the virus must be capable of replicating in the presence of maternally-derived antibodies against the virus, without being pathogenic to the embryo. Current IBV vaccines are derived following multiple passage in embryonated eggs, this results in viruses with reduced pathogenicity for chickens, so that they can be used as live attenuated vaccines. However such viruses almost always show an increased virulence to embryos and therefore cannot be used for in ova vaccination as they cause reduced hatchability. A 70% reduction in hatchability is seen in some cases.

Attenuation following multiple passage in embryonated eggs also suffers from other disadvantages. It is an empirical method, as attenuation of the viruses is random and will differ every time the virus is passaged, so passage of the same virus through a different series of eggs for attenuation purposes will lead to a different set of mutations leading to attenuation. There are also efficacy problems associated with the process: some mutations will affect the replication of the virus and some of the mutations may make the virus too attenuated. Mutations can also occur in the S gene which may also affect immunogenicity so that the desired immune response is affected and the potential vaccine may not protect against the required serotype. In addition there are problems associated with reversion to virulence and stability of vaccines.

It is important that new and safer vaccines are developed for the control of IBV. Thus there is a need for IBV vaccines which are not associated with these issues, in particular vaccines which may be used for in ovo vaccination.
SUMMARY OF ASPECTS OF THE INVENTION
The present inventors have used a reverse genetics approach in order to rationally attenuate IBV. This approach is much more controllable than random attenuation following multiple passages in embryonated eggs because the position of each mutation is known and its effect on the virus, i.e. the reason for attenuation, can be derived.

Using their reverse genetics approach, the present inventors have identified various mutations which cause the virus to have reduced levels of pathogenicity. The levels of pathogenicity may be reduced such that when the virus is administered to an embryonated egg, it is capable of replicating without being pathogenic to the embryo. Such viruses may be suitable for in ovo vaccination, which is a significant advantage and has improvement over attenuated IBV vaccines produced following multiple passage in embryonated eggs.

Thus in a first aspect, the present invention provides a live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or more of non-structural protein(s) (nsp)-10, nsp-14, nsp-15 or nsp-16.

The variant replicase gene may encode a protein comprising one or more amino acid mutations selected from the list of:
    • Pro to Leu at position 85 of SEQ ID NO: 6,
    • Val to Leu at position 393 of SEQ ID NO: 7;
    • Leu to Ile at position 183 of SEQ ID NO: 8;
    • Val to Ile at position 209 of SEQ ID NO: 9.

The replicase gene may encode a protein comprising the amino acid mutation Pro to Leu at position 85 of SEQ ID NO: 6.

The replicase gene may encode a protein comprising the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183 of SEQ ID NO: 8; and Val to Ile at position 209 of SEQ ID NO: 9.

The replicase gene may encodes a protein comprising the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6; Val to Leu at position 393 of SEQ ID NO:7; Leu to Ile at position 183 of SEQ ID NO:8; and Val to Ile at position 209 of SEQ ID NO: 9.

The replicase gene may comprise one or more nucleotide substitutions selected from the list of:

C to T at nucleotide position 12137;

G to C at nucleotide position 18114;

T to A at nucleotide position 19047; and

G to A at nucleotide position 20139;

compared to the sequence shown as SEQ ID NO: 1.

The coronavirus may be an infectious bronchitis virus (IBV).

The coronavirus may be IBV M41.

The coronavirus may comprise an S protein at least part of which is from an IBV serotype other than M41.

For example, the S1 subunit or the entire S protein may be from an IBV serotype other than M41.

The coronavirus according to the first aspect of the invention has reduced pathogenicity compared to a coronavirus expressing a corresponding wild-type replicase, such that when the virus is administered to an embryonated egg, it is capable of replicating without being pathogenic to the embryo.

In a second aspect, the present invention provides a variant replicase gene as defined in connection with the first aspect of the invention.

In a third aspect, the present invention provides a protein encoded by a variant coronavirus replicase gene according to the second aspect of the invention.

In a fourth aspect, the present invention provides a plasmid comprising a replicase gene according to the second aspect of the invention.

In a fifth aspect, the present invention provides a method for making the coronavirus according to the first aspect of the invention which comprises the following steps:
    • (i) transfecting a plasmid according to the fourth aspect of the invention into a host cell;
    • (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene;
    • (iii) allowing homologous recombination to occur between the replicase gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified replicase gene; and
    • (iv) selecting for recombining virus comprising the modified replicase gene.

The recombining virus may be a vaccinia virus.

The method may also include the step:
    • (v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).

In a sixth aspect, the present invention provides a cell capable of producing a coronavirus according to the first aspect of the invention.

In a seventh aspect, the present invention provides a vaccine comprising a coronavirus according to the first aspect of the invention and a pharmaceutically acceptable carrier.

In an eighth aspect, the present invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to the seventh aspect of the invention to the subject.

Further aspects of the invention provide:
    • the vaccine according to the seventh aspect of the invention for use in treating and/or preventing a disease in a subject.
    • use of a coronavirus according to the first aspect of the invention in the manufacture of a vaccine for treating and/or preventing a disease in a subject.

The disease may be infectious bronchitis (IB).

The method of administration of the vaccine may be selected from the group consisting of; eye drop administration, intranasal administration, drinking water administration, post-hatch injection and in ovo injection.

Vaccination may be by in ova vaccination.

The present invention also provides a method for producing a vaccine according to the seventh aspect of the invention, which comprises the step of infecting a cell according to the sixth aspect of the invention with a coronavirus according to the first aspect of the invention.
DESCRIPTION OF THE FIGURES
FIG. 1—Growth kinetics of M41-R-6 and M41-R-12 compared to M41-CK (M41 EP4) on CK cells

FIG. 2—Clinical signs, snicking and wheezing, associated with M41-R-6 and M41-R-12 compared to M41-CK (M41 EP4) and Beau-R (Bars show mock, Beau-R, M41-R 6, M41-R 12, M41-CK EP4 from left to right of each timepoint).

FIG. 3—Ciliary activity of the viruses in tracheal rings isolated from tracheas taken from infected chicks. 100% ciliary activity indicates no effect by the virus; apathogenic, 0% activity indicates complete loss of ciliary activity, complete ciliostasis, indicating the virus is pathogenic (Bars show mock, Beau-R, M41-R 6, M41-R 12, M41-CK EP4 from left to right of each timepoint).

FIG. 4—Clinical signs, snicking, associated with M41R-nsp10rep and M41R-nsp14,15,16rep compared to M41-R-12 and M41-CK (M41 EP5) (Bars show mock, M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 from left to right of each timepoint).

FIG. 5—Ciliary activity of M41R-nsp10rep and M41R-nsp14,15,16rep compared to M41-R-12 and M41-CK in tracheal rings isolated from tracheas taken from infected chicks (Bars show mock; M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 from left to right of each timepoint).

FIG. 6—Clinical signs, snicking, associated with M41R-nsp10, 15rep, M41R-nsp10, 14, 15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared to M41-CK (Bars show mock, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from left to right of each timepoint).

FIG. 7—Clinical signs, wheezing, associated with M41R-nsp10, 15rep, M41R-nsp10, 14, 15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared to M41-CK (Bars show mock, M41R-nsp10,15rep1; M14R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from left to right of each timepoint).

FIG. 8—Ciliary activity of M41R-nsp10, 15rep, M41R-nsp10, 14, 15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared to M41-CK in tracheal rings isolated from tracheas taken from infected chicks (Bars show mock, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from left to right of each timepoint).

FIG. 9—Growth kinetics of rIBVs compared to M41-CK on CK cells. FIG. 9A shows the results for M41-R and M41-K. FIG. 9B shows the results for M41-nsp10 rep; M41R-nsp14, 15, 16 rep; M41R-nsp10, 15 rep; M41R-nsp10, 15, 16 rep; M41R-nsp10, 14, 15 rep; and M41R-nsp10, 14, 16.

FIG. 10—Position of amino acid mutations in mutated nsp10, nsp14, nsp15 and nsp16 sequences.

FIG. 11—A) Snicking; B) Respiratory symptoms (wheezing and rales combined) and C) Ciliary activity of rIBV M41R-nsp 10,14 rep and rIBV M41R-nsp 10,16 rep compared to M41-CK (Bars show mock, M41R-nsp10,14rep; M41R-nsp10,16rep and M41-K from left to right of each timepoint).
DETAILED DESCRIPTION
The present invention provides a coronavirus comprising a variant replicase gene which, when expressed in the coronavirus, causes the virus to have reduced pathogenicity compared to a corresponding coronavirus which comprises the wild-type replicase gene.

Coronavirus

Gammacoronavirus is a genus of animal virus belonging to the family Coronaviridae. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical symmetry.

The genomic size of coronaviruses ranges from approximately 27 to 32 kilobases, which is the longest size for any known RNA virus.

Coronaviruses primarily infect the upper respiratory or gastrointestinal tract of mammals and birds. Five to six different currently known strains of coronaviruses infect humans. The most publicized human coronavirus, SARS-CoV which causes severe acute respiratory syndrome (SARS), has a unique pathogenesis because it causes both upper and lower respiratory tract infections and can also cause gastroenteritis. Middle East respiratory syndrome coronavirus (MERS-CoV) also causes a lower respiratory tract infection in humans. Coronaviruses are believed to cause a significant percentage of all common colds in human adults.

Coronaviruses also cause a range of diseases in livestock animals and domesticated pets, some of which can be serious and are a threat to the farming industry. Economically significant coronaviruses of livestock animals include infectious bronchitis virus (IBV) which mainly causes respiratory disease in chickens and seriously affects the poultry industry worldwide; porcine coronavirus (transmissible gastroenteritis, TGE) and bovine coronavirus, which both result in diarrhoea in young animals. Feline coronavirus has two forms, feline enteric coronavirus is a pathogen of minor clinical significance, but spontaneous mutation of this virus can result in feline infectious peritonitis (FIP), a disease associated with high mortality.

There are also two types of canine coronavirus (CCoV), one that causes mild gastrointestinal disease and one that has been found to cause respiratory disease. Mouse hepatitis virus (MHV) is a coronavirus that causes an epidemic murine illness with high mortality, especially among colonies of laboratory mice.

Coronaviruses are divided into four groups, as shown below:
    • Alpha
      • Canine coronavirus (CCoV)
      • Feline coronavirus (FeCoV)
      • Human coronavirus 229E (HCoV-229E)
      • Porcine epidemic diarrhoea virus (PEDV)
      • Transmissible gastroenteritis virus (TGEV)
      • Human Coronavirus NL63 (NL or New Haven)
    • Beta
      • Bovine coronavirus (BCoV)
      • Canine respiratory coronavirus (CRCoV)—Common in SE Asia and Micronesia
      • Human coronavirus OC43 (HCoV-OC43)
      • Mouse hepatitis virus (MHV)
      • Porcine haemagglutinating encephalomyelitis virus (HEV)
      • Rat coronavirus (Roy). Rat Coronavirus is quite prevalent in Eastern Australia where, as of March/April 2008, it has been found among native and feral rodent colonies.
      • (No common name as of yet) (HCoV-HKU1)
      •  Severe acute respiratory syndrome coronavirus (SARS-CoV)
      • Middle East respiratory syndrome coronavirus (MERS-CoV)
    • Gamma
      • Infectious bronchitis virus (IBV)
      • Turkey coronavirus (Bluecomb disease virus)
      • Pheasant coronavirus
      • Guinea fowl coronavirus
    • Delta
      • Bulbul coronavirus (BuCoV)
      • Thrush coronavirus (ThCoV)
      • Munia coronavirus (MuCoV)
      • Porcine coronavirus (PorCov) HKU15

The variant replicase gene of the coronavirus of the present invention may be derived from an alphacoronavirus such as TGEV; a betacoronavirus such as MHV; or a gammacoronavirus such as IBV.

As used herein the term “derived from” means that the replicase gene comprises substantially the same nucleotide sequence as the wild-type replicase gene of the relevant coronavirus. For example, the variant replicase gene of the present invention may have up to 80%, 85%, 90%, 95%, 98% or 99% identity with the wild type replicase sequence. The variant coronavirus replicase gene encodes a protein comprising a mutation in one or more of non-structural protein (nsp)-10, nsp-14, nsp-15 or nsp-16 when compared to the wild-type sequence of the non-structural protein.

IBV

Avian infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens which causes significant economic losses. The disease is characterized by respiratory signs including gasping, coughing, sneezing, tracheal rales, and nasal discharge. In young chickens, severe respiratory distress may occur. In layers, respiratory distress, nephritis, decrease in egg production, and loss of internal egg quality and egg shell quality are common.

In broilers, coughing and rattling are common clinical signs, rapidly spreading in all the birds of the premises. Morbidity is 100% in non-vaccinated flocks. Mortality varies depending on age, virus strain, and secondary infections but may be up to 60% in non-vaccinated flocks.

The first IBV serotype to be identified was Massachusetts, but in the United States several serotypes, including Arkansas and Delaware, are currently circulating, in addition to the originally identified Massachusetts type.

The IBV strain Beaudette was derived following at least 150 passages in chick embryos. IBV Beaudette is no longer pathogenic for hatched chickens but rapidly kills embryos.

H120 is a commercial live attenuated IBV Massachusetts serotype vaccine strain, attenuated by approximately 120 passages in embryonated chicken eggs. H52 is another Massachusetts vaccine, and represents an earlier and slightly more pathogenic passage virus (passage 52) during the development of H120. Vaccines based on H120 are commonly used.

IB QX is a virulent field isolate of IBV. It is sometimes known as “Chinese QX” as it was originally isolated following outbreaks of disease in the Qingdao region in China in the mid 1990s. Since that time the virus has crept towards Europe. From 2004, severe egg production issues have been identified with a very similar virus in parts of Western Europe, predominantly in the Netherlands, but also reported from Germany, France, Belgium, Denmark and in the UK.

The virus isolated from the Dutch cases was identified by the Dutch Research Institute at Deventer as a new strain that they called D388. The Chinese connection came from further tests which showed that the virus was 99% similar to the Chinese QX viruses. A live attenuated QX-like IBV vaccine strain has now been developed.

IBV is an enveloped virus that replicates in the cell cytoplasm and contains an non-segmented, single-stranded, positive sense RNA genome. IBV has a 27.6 kb RNA genome and like all coronaviruses contains the four structural proteins; spike glycoprotein (S), small membrane protein (E), integral membrane protein (M) and nucleocapsid protein (N) which interacts with the genomic RNA.

The genome is organised in the following manner: 5′UTR—polymerase (replicase) gene—structural protein genes (S-E-M-N)—UTR 3′; where the UTR are untranslated regions (each ˜500 nucleotides in IBV).

The lipid envelope contains three membrane proteins: S, M and E. The IBV S protein is a type I glycoprotein which oligomerizes in the endoplasmic reticulum and is assembled into homotrimer inserted in the virion membrane via the transmembrane domain and is associated through non-covalent interactions with the M protein. Following incorporation into coronavirus particles, the S protein is responsible for binding to the target cell receptor and fusion of the viral and cellular membranes. The S glycoprotein consists of four domains: a signal sequence that is cleaved during synthesis; the ectodomain, which is present on the outside of the virion particle; the transmembrane region responsible for anchoring the S protein into the lipid bilayer of the virion particle; and the cytoplasmic tail.

All coronaviruses also encode a set of accessory protein genes of unknown function that are not required for replication in vitro, but may play a role in pathogenesis. IBV encodes two accessory genes, genes 3 and 5, which both express two accessory proteins 3a, 3b and 5a, 5b, respectively.

The variant replicase gene of the coronavirus of the present invention may be derived from an IBV. For example the IBV may be IBV Beaudette, H120, H52, IB QX, D388 or M41.

The IBV may be IBV M41. M41 is a prototypic Massachusetts serotype that was isolated in the USA in 1941. It is an isolate used in many labs throughout the world as a pathogenic lab stain and can be obtained from ATCC (VR-21™). Attenuated variants are also used by several vaccine producers as IBV vaccines against Massachusetts serotypes causing problems in the field. The present inventors chose to use this strain as they had worked for many years on this virus, and because the sequence of the complete virus genome is available. The M41 isolate, M41-CK, used by the present inventors was adapted to grow in primary chick kidney (CK) cells and was therefore deemed amenable for recovery as an infectious virus from a cDNA of the complete genome. It is representative of a pathogenic IBV and therefore can be analysed for mutations that cause either loss or reduction in pathogenicity.

The genome sequence of IBV M41-CK is provided as SEQ ID NO: 1.

IBV M41-CK Sequence 
Reduced Pathogenicity

The live, attenuated coronavirus of the present invention comprises a variant replicase gene which causes the virus to have reduced pathogenicity compared to a coronavirus expressing the corresponding wild-type gene.

The term “attenuated” as used herein, refers to a virus that exhibits said reduced pathogenicity and may be classified as non-virulent. A live, attenuated virus is a weakened replicating virus still capable of stimulating an immune response and producing immunity but not causing the actual illness.

The term “pathogenicity” is used herein according to its normal meaning to refer to the potential of the virus to cause disease in a subject. Typically the pathogenicity of a coronavirus is determined by assaying disease associated symptoms, for example sneezing, snicking and reduction in tracheal ciliary activity.

The term “reduced pathogenicity” is used to describe that the level of pathogenicity of a coronavirus is decreased, lessened or diminished compared to a corresponding, wild-type coronavirus.

In one embodiment, the coronavirus of the present invention has a reduced pathogenicity compared to the parental M41-CK virus from which it was derived or a control coronavirus. The control coronavirus may be a coronavirus with a known pathogenicity, for example a coronavirus expressing the wild-type replicase protein.

The pathogenicity of a coronavirus may be assessed utilising methods well-known in the art. Typically, pathogenicity is assessed by assaying clinical symptoms in a subject challenged with the virus, for example a chicken.

As an illustration, the chicken may be challenged at 8-24 days old by nasal or ocular inoculation. Clinical symptoms, associated with IBV infection, may be assessed 3-10 days post-infection. Clinical symptoms commonly assessed to determine the pathogenicity of a coronavirus, for example an IBV, include gasping, coughing, sneezing, snicking, depression, ruffled feathers and loss of tracheal ciliary activity.

The variant replicase of the present invention, when expressed in a coronavirus, may cause a reduced level of clinical symptoms compared to a coronavirus expressing a wild-type replicase.

For example a coronavirus expressing the variant replicase may cause a number of snicks per bird per minute which is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the number of snicks caused by a virus expressing the wild type replicase.

A coronavirus expressing a variant replicase according to the present invention may cause wheezing in less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the number of birds in a flock infected with the a virus expressing the wild type replicase.

A coronavirus expressing a variant replicase according to the present invention may result in tracheal ciliary activity which is at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the level of tracheal ciliary activity in uninfected birds.

A coronavirus expressing a variant replicase according to the present invention may cause clinical symptoms, as defined in Table 2, at a lower level than a coronavirus expressing the wild type replicase.

TABLE 2 IBV severity limits based on clinical signs:

The variant replicase of the present invention, when expressed in a coronavirus, may cause the virus to replicate at non-pathogenic levels in ovo.

While developing vaccines to be administered in ovo to chicken embryos, attention must be paid to two points: the effect of maternal antibodies on the vaccines and the effect of the vaccines on the embryo. Maternal antibodies are known to interfere with active immunization. For example, vaccines with mild strains do not induce protective antibody levels when administered to broiler chickens with maternal antibodies as these strains are neutralized by the maternal antibody pool.

Thus a viral particle must be sufficiently efficient at replicating and propagating to ensure that it is not neutralized by the maternally-derived antibodies against the virus. Maternally-derived antibodies are a finite pool of effective antibodies, which decrease as the chicken ages, and neutralization of the virus in this manner does not equate to the establishment of long-term immunity for the embryo/chick. In order to develop long-term immunity against the virus, the embryo and hatched chicken must develop an appropriate protective immune response which is distinct to the effect of the maternally-derived antibodies.

To be useful for in ovo vaccination, the virus must also not replicate and propagate at a level which causes it to be pathogenic to the embryo.

Reduced pathogenicity in terms of the embryo may mean that the coronavirus causes less reduction in hatchability compared to a corresponding, wild-type control coronavirus. Thus the term “without being pathogenic to the embryo” in the context of the present invention may mean “without causing reduced hatchability” when compared to a control coronavirus.

A suitable variant replicase may be identified using methods which are known in the art. For example comparative challenge experiments following in ovo vaccination of embryos with or without maternally-derived antibodies may be performed (i.e. wherein the layer has or has not been vaccinated against IBV).

If the variant replicase enables the virus to propagate at a level which is too high, the embryo will not hatch or will not be viable following hatching (i.e. the virus is pathogenic to the embryo). A virus which is pathogenic to the embryo may kill the embryo.

If the variant replicase causes a reduction in viral replication and propagation which is too great, the virus will be neutralised by the maternally-derived antibodies. Subsequent challenge of the chick with IBV will therefore result in the development of clinical symptoms (for example wheezing, snicking, loss of ciliary activity) and the onset of disease in the challenged chick; as it will have failed to develop effective immunity against the virus.

Variant

As used herein, the term ‘variant’ is synonymous with ‘mutant’ and refers to a nucleic acid or amino acid sequence which differs in comparison to the corresponding wild-type sequence.

A variant/mutant sequence may arise naturally, or may be created artificially (for example by site-directed mutagenesis). The mutant may have at least 70, 80, 90, 95, 98 or 99% sequence identity with the corresponding portion of the wild type sequence. The mutant may have less than 20, 10, 5, 4, 3, 2 or 1 mutation(s) over the corresponding portion of the wild-type sequence.

The term “wild type” is used to mean a gene or protein having a nucleotide or amino acid sequence which is identical with the native gene or protein respectively (i.e. the viral gene or protein).

Identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % identity between two or more sequences. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools, ClustalX (see Larkin et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestf it program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).

The sequence may have one or more deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent molecule. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the activity is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

ALIPHATIC Non-polar G A P I L V Polar- uncharged C S T M N Q Polar- charged D E K R AROMATIC H F W Y

The coronavirus of the present invention may comprise a variant replicase gene which encodes a protein which comprises a mutation compared to any one of SEQ ID NO: 6, 7, 8 or 9 which, when expressed in a coronavirus, causes the virus to have reduced pathogenicity compared to a coronavirus expressing the corresponding wild-type replicase.

The variant replicase gene may encode a protein which comprises at least one or more amino acid mutations in any combination of nsp-10, nsp-14, nsp-15 and nsp-16.

The variant replicase gene of the coronavirus of the present invention may encode a protein comprising a mutation as defined in the M41 mod sequences presented in FIG. 10.

The variant replicase gene of the coronavirus of the present invention may encode a protein which comprises one or more amino acid mutations selected from the list of:
    • Pro to Leu at position 85 of SEQ ID NO: 6,
    • Val to Leu at position 393 of SEQ ID NO: 7;
    • Leu to Ile at position 183 of SEQ ID NO: 8;
    • Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene of the coronavirus of the present invention may encode a protein which does not comprise a mutation in nsp-2, nsp-3, nsp-6 or nsp-13.

The variant replicase gene of the coronavirus of the present invention may encode a protein which does not comprise a mutation in nsp10 which corresponds to the threonine to isoleucine mutation caused by a mutation at nucleotide position 12,008 in the gene reported by Ammayappan et al. (Arch Virol (2009) 154:495-499).

Ammayappan et al (as above) reports the identification of sequence changes responsible for the attenuation of IBV strain Arkansas DPI. The study identified 17 amino acid changes in a variety of IBV proteins following multiple passages, approx. 100, of the virus in embryonated eggs. It was not investigated whether the attenuated virus (Ark DPI 101) is capable of replicating in the presence of maternally-derived antibodies against the virus in ovo, without being pathogenic to the embryo. Given that this virus was produced by multiple passage in SPF embryonated eggs, similar methodology for classical IBV vaccines, it is likely that this virus is pathogenic for embryos. The virus may also be sensitive to maternally-derived antibodies if the hens were vaccinated with a similar serotype.

The variant replicase gene of the coronavirus of the present invention may encode a protein which comprises any combination of one or more amino acid mutations provided in the list above.

The variant replicase gene may encode a protein which comprises the amino acid mutation Pro to Leu at position 85 of SEQ ID NO: 6.

The variant replicase gene may encode a protein which comprises the amino acid mutation Val to Leu at position 393 of SEQ ID NO: 7.

The variant replicase gene may encode a protein which comprises the amino acid mutation Leu to Ile at position 183 of SEQ ID NO: 8.

The variant replicase gene may encode a protein which comprises the amino acid mutation Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, and Val to Leu at position 393 of SEQ ID NO: 7.

The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 Leu to Ile at position 183 of SEQ ID NO: 8.

The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 and Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene may encode a protein which comprises the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7 and Leu to Ile at position 183 of SEQ ID NO: 8.

The variant replicase gene may encode a protein which comprises the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7 and Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene may encode a protein which comprises the amino acid mutations Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7 and Leu to Ile at position 183 of SEQ ID NO: 8.

The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7 and Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene may encode a protein which comprises the amino acid mutations Val to Leu at position 393 of SEQ ID NO: 7, Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene may encode a protein which comprises the amino acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 of SEQ ID NO: 7, Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at position 209 of SEQ ID NO: 9.

The variant replicase gene may also be defined at the nucleotide level.

For example the nucleotide sequence of the variant replicase gene of the coronavirus of the present invention may comprise one or more nucleotide substitutions within the regions selected from the list of: 11884-12318, 16938-18500, 18501-19514 and 19515-20423 of SEQ ID NO:1.

For example the nucleotide sequence of the variant replicase gene of the coronavirus of the present invention may comprise one or more nucleotide substitutions selected from the list of:
    • C to Tat nucleotide position 12137;
    • G to C at nucleotide position 18114;
    • T to A at nucleotide position 19047; and
    • G to A at nucleotide position 20139;
      compared to the sequence shown as SEQ ID NO: 1.

As used herein, the term “substitution” is synonymous with the term mutation and means that the nucleotide at the identified position differs to that of the wild-type nucleotide sequence.

The nucleotide sequence may comprise any combination of the nucleotide substitutions selected from the list of:
    • C to Tat nucleotide position 12137;
    • G to Cat nucleotide position 18114;
    • T to A at nucleotide position 19047; and
    • G to A at nucleotide position 20139;
      compared to the sequence shown as SEQ ID NO: 1.

The nucleotide sequence may comprise the substitution C12137T.

The nucleotide sequence may comprise substitution G18114C.

The nucleotide sequence may comprise the substitution T19047A.

The nucleotide sequence may comprise the substitution G20139A.

The nucleotide sequence may comprise the substitutions C12137T and G18114C.

The nucleotide sequence may comprise the substitutions C12137T and T19047A.

The nucleotide sequence may comprise the substitutions C12137T and G20139A.

The nucleotide sequence may comprise the substitutions G18114C and T19047A.

The nucleotide sequence may comprise the substitutions G18114C and G20139A.

The nucleotide sequence may comprise the substitutions T19047A and G20139A.

The nucleotide sequence may comprise the substitutions C12137T, G18114C and T19047A.

The nucleotide sequence may comprise the substitutions C12137T, T19047A and G20139A.

The nucleotide sequence may comprise the substitutions C12137T, G18114C and G20139A.

The nucleotide sequence may comprise the substitutions G18114C, T19047A and G20139A.

The nucleotide sequence may comprise the substitutions C12137T, G18114C, T19047A and G20139A.

The nucleotide sequence may not comprise a substitution which corresponds to the C12008T substitution reported by Ammayappan et al. (as above).

The nucleotide sequence may be natural, synthetic or recombinant. It may be double or single stranded, it may be DNA or RNA or combinations thereof. It may, for example, be cDNA, PCR product, genomic sequence or mRNA.

The nucleotide sequence may be codon optimised for production in the host/host cell of choice.

It may be isolated, or as part of a plasmid, virus or host cell.

Plasmid

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. They are usually circular and double-stranded.

Plasmids, or vectors (as they are sometimes known), may be used to express a protein in a host cell. For example a bacterial host cell may be transfected with a plasmid capable of encoding a particular protein, in order to express that protein. The term also includes yeast artificial chromosomes and bacterial artificial chromosomes which are capable of accommodating longer portions of DNA.

The plasmid of the present invention comprises a nucleotide sequence capable of encoding a defined region of the replicase protein. It may also comprise one or more additional coronavirus nucleotide sequence(s), or nucleotide sequence(s) capable of encoding one or more other coronavirus proteins such as the S gene and/or gene 3.

The plasmid may also comprise a resistance marker, such as the guanine xanthine phosphoribosyltransferase gene (gpt) from Escherichia coli, which confers resistance to mycophenolic acid (MPA) in the presence of xanthine and hypoxanthine and is controlled by the vaccinia virus P7.5 early/late promoter.

Recombinant Vaccinia Virus

The present invention also relates to a recombinant vaccinia virus (rVV) comprising a variant replicase gene as defined herein.

The recombinant vaccinia virus (rVV) may be made using a vaccinia-virus based reverse genetics system.

In this respect, the present invention also provides a method for making a viral particle by:
    • (i) transfecting a plasmid as described in the previous section into a host cell;
    • (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene;
    • (iii) allowing homologous recombination to occur between the replicase gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified replicase gene;
    • (iv) selecting for recombining virus comprising the modified replicase gene.

The term ‘modified replicase gene’ refers to a replicase gene which comprises a variant replicase gene as described in connection with the first aspect of the present invention. Specifically, the term refers to a gene which is derived from a wild-type replicase gene but comprises a nucleotide sequence which causes it to encode a variant replicase protein as defined herein.

The recombination may involve all or part of the replicase gene. For example the recombination may involve a nucleotide sequence encoding for any combination of nsp-10, nsp-14, nsp-15 and/or nsp-16. The recombination may involve a nucleotide sequence which encodes for an amino acid mutation or comprises a nucleotide substitution as defined above.

The genome of the coronavirus strain may lack the part of the replicase protein corresponding to the part provided by the plasmid, so that a modified protein is formed through insertion of the nucleotide sequence provided by the plasmid.

The recombining virus is one suitable to allow homologous recombination between its genome and the plasmid. The vaccinia virus is particularly suitable as homologous recombination is routinely used to insert and delete sequences for the vaccinia virus genome.

The above method optionally includes the step:
    • (v) recovery of recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).

Methods for recovering recombinant coronavirus, such as recombinant IBV, are known in the art (See Britton et al (2005) see page 24; and PCT/GB2010/001293).

For example, the DNA from the recombining virus from step (iv) may be inserted into a plasmid and used to transfect cells which express cytoplasmic T7 RNA polymerase. The cells may, for example be pre-infected with a fowlpox virus expressing T7 RNA polymerase. Recombinant coronavirus may then be isolated, for example, from the growth medium.

When the plasmid is inserted into the vaccinia virus genome, an unstable intermediate is formed. Recombinants comprising the plasmid may be selected for e.g. using a resistance marker on the plasmid.

Positive recombinants may then be verified to contain the modified replicase gene by, for example, PCR and sequencing.

Large stocks of the recombining virus including the modified replicase gene (e.g. recombinant vaccinia virus, (rVV) may be grown up and the DNA extracted in order to carry out step (v)).

Suitable reverse genetics systems are known in the art (Casais et al (2001) J. Virol 75:12359-12369; Casais et al (2003) J. Virol. 77:9084-9089; Britton et al (2005) J. Virological Methods 123:203-211; Armesto et al (2008) Methods in Molecular Biology 454:255-273).

Cell

The coronavirus may be used to infect a cell.

Coronavirus particles may be harvested, for example from the supernatant, by methods known in the art, and optionally purified.

The cell may be used to produce the coronavirus particle.

Thus the present invention also provides a method for producing a coronavirus which comprises the following steps:

(i) infection of a cell with a coronavirus according to the invention;

(ii) allowing the virus to replicate in the cell; and

(iii) harvesting the progeny virus.

The present invention also provides a cell capable of producing a coronavirus according to the invention using a reverse genetics system. For example, the cell may comprise a recombining virus genome comprising a nucleotide sequence capable of encoding the replicase gene of the present invention.

The cell may be able to produce recombinant recombining virus (e.g. vaccinia virus) containing the replicase gene.

Alternatively the cell may be capable of producing recombinant coronavirus by a reverse genetics system. The cell may express or be induced to express T7 polymerase in order to rescue the recombinant viral particle.

Vaccine

The coronavirus may be used to produce a vaccine. The vaccine may by a live attenuated form of the coronavirus of the present invention and may further comprise a pharmaceutically acceptable carrier. As defined herein, “pharmaceutically acceptable carriers” suitable for use in the invention are well known to those of skill in the art. Such carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcohol/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusting agents may also be employed. Buffers include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, histidine-Hel, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Iris, trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EDTA), inert gases and the like may also be provided in the pharmaceutical carriers. The present invention is not limited by the selection of the carrier. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art. See, e.g., texts such as Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, pub!., 2000; and The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds. R. C. Rowe et al, APhA Publications, 2003.

The vaccine of the invention will be administered in a “therapeutically effective amount”, which refers to an amount of an active ingredient, e.g., an agent according to the invention, sufficient to effect beneficial or desired results when administered to a subject or patient. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition according to the invention may be readily determined by one of ordinary skill in the art. In the context of this invention, a “therapeutically effective amount” is one that produces an objectively measured change in one or more parameters associated Infectious Bronchitis condition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to reduce the incidence of Infectious Bronchitis. As used herein, the term “therapeutic” encompasses the full spectrum of treatments for a disease, condition or disorder. A “therapeutic” agent of the invention may act in a manner that is prophylactic or preventive, including those that incorporate procedures designed to target animals that can be identified as being at risk (pharmacogenetics); or in a manner that is ameliorative or curative in nature; or may act to slow the rate or extent of the progression of at least one symptom of a disease or disorder being treated.

The present invention also relates to a method for producing such a vaccine which comprises the step of infecting cells, for example Vero cells, with a viral particle comprising a replicase protein as defined in connection with the first aspect of the invention.

Vaccination Method

The coronavirus of the present invention may be used to treat and/or prevent a disease.

To “treat” means to administer the vaccine to a subject having an existing disease in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

To “prevent” means to administer the vaccine to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease (e.g. infection) or to reduce or prevent development of at least one symptom associated with the disease.

The disease may be any disease caused by a coronavirus, such as a respiratory disease and and/or gastroenteritis in humans and hepatitis, gastroenteritis, encephalitis, or a respiratory disease in other animals.

The disease may be infectious bronchitis (IB); Porcine epidemic diarrhoea; Transmissible gastroenteritis; Mouse hepatitis virus; Porcine haemagglutinating encephalomyelitis; Severe acute respiratory syndrome (SARS); or Bluecomb disease.

The disease may be infectious bronchitis.

The vaccine may be administered to hatched chicks or chickens, for example by eye drop or intranasal administration. Although accurate, these methods can be expensive e.g. for large broiler flocks. Alternatives include spray inoculation of administration to drinking water but it can be difficult to ensure uniform vaccine application using such methods.

The vaccine may be provided in a form suitable for its administration, such as an eye-dropper for intra-ocular use.

The vaccine may be administered by in ovo inoculation, for example by injection of embryonated eggs. In ovo vaccination has the advantage that it provides an early stage resistance to the disease. It also facilitates the administration of a uniform dose per subject, unlike spray inoculation and administration via drinking water.

The vaccine may be administered to any suitable compartment of the egg, including allantoic fluid, yolk sac, amnion, air cell or embryo. It may be administered below the shell (aircell) membrane and chorioallantoic membrane.

Usually the vaccine is injected into embryonated eggs during late stages of embryonic development, generally during the final quarter of the incubation period, such as 3-4 days prior to hatch. In chickens, the vaccine may be administered between day 15-19 of the 21-day incubation period, for example at day 17 or 18.

The process can be automated using a robotic injection process, such as those described in WO 2004/078203.

The vaccine may be administered together with one or more other vaccines, for example, vaccines for other diseases, such as Newcastle disease virus (NDV). The present invention also provides a vaccine composition comprising a vaccine according to the invention together with one or more other vaccine(s). The present invention also provides a kit comprising a vaccine according to the invention together with one or more other vaccine(s) for separate, sequential or simultaneous administration.

The vaccine or vaccine composition of the invention may be used to treat a human, animal or avian subject. For example, the subject may be a chick, chicken or mouse (such as a laboratory mouse, e.g. transgenic mouse).

Typically, a physician or veterinarian will determine the actual dosage which will be most suitable for an individual subject or group of subjects and it will vary with the age, weight and response of the particular subject(s).

The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the delivery or immunogenicity of the virus.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

https://patents.justia.com/patent/10130701