Melinda Gates wants to target black people for priority covid-19 vaccines

Monday, July 06, 2020 by: Ethan Huff

(Natural News) During a recent interview with TIME, Melinda Gates, co-chair of the infamous Bill & Melinda Gates Foundation, spelled out the order in which she believes people need to be vaccinated for the Wuhan coronavirus (COVID-19). First in line after health care workers, she divulged, will be black people, which are supposedly more at risk of infection than others.

In order to achieve “health equity,” black people will need to be given first priority for the jabs, which are being rushed to market as quickly as possible, likely before the end of the year.

Using the George Floyd saga as the impetus behind why Wuhan coronavirus (COVID-19) vaccines need to be administered to black people first, Gates is encouraging the public to “pause during this time and learn as best we can from it.” And to her this means lining up the blacks to be injected with Wuhan coronavirus (COVID-19) vaccines before anyone else gets them.

“Even before we saw this senseless death, COVID had already started to show us gaps and structural problems in our country,” Gates is quoted as saying. “We are seeing black men die at a disproportionate rate. We know the way out of COVID-19 will be a vaccine, and it needs to go out equitably.”

Listen below to The Health Ranger Report as Mike Adams, the Health Ranger, talks about how the vaccine industry could end up destroying itself by rushing out a Wuhan coronavirus (COVID-19) vaccine:

Bill and Melinda Gates have been planning for this moment for 20 years

When asked how she plans to bring these plans to fruition, Gates explained that she and her husband have been greasing the wheels for more than 20 years. They set up an advanced vaccine delivery system to be used for such a time as this, and they have been working with governments to purchase vaccines in bulk for rapid distribution.

Both Bill and Melinda Gates have essentially been getting governments and other power players all around the world to sign on to a pledge stating, “We care about this vaccine getting out equitably.” And by donating to GAVI all these years, they have been effectively contributing towards the rapid push for a Wuhan coronavirus (COVID-19) vaccine.

“The first people that need this vaccine are the 60 million health care workers around the world,” Gates further stated. “They deserve to get it before anybody else. Then you start tiering.”

“In the U.S., that would be black people next, quite honestly, and many other people of color,” she added. “They are having disproportionate effects from COVID-19. From there, people with underlying health conditions, and then people who are older. Those are the ones who all need it first.”

Recognizing that the Gates family lineage is packed with eugenicists, it is hardly surprising that the “undesirables” all come first with this thing. And as for the health care workers, with them out of the way there will be no more health care system to take care of anyone.

Gates also wants to make sure that other “essential” workers are also jabbed first, including the people “who are keeping our grocery stores open for us so we can buy food, or who are making sure that food moves through the warehouses.”

To those who believe that vaccines are good and that one for the Wuhan coronavirus (COVID-19) will help society, this all sounds very noble. But to those who see vaccines as biological weapons, the plan to vaccinate everyone “important” and “vulnerable” first reveals a much darker agenda at play. (Click to Source)

For more related news about the dangers of vaccination, be sure to check out Vaccines.news.

Sources for this article include:

TIME.com

NaturalNews.com

 

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Protesters in South Africa burn masks in opposition to coronavirus vaccine experimentation

Monday, July 06, 2020 by: Ethan Huff

(Natural News) Africans are finally waking up to the deception of the experimental vaccination campaigns that continue to be thrust upon them in the name of advancing “public health,” and the globalist overlords behind it all are beginning to quake in their boots.

A recent protest in South Africa saw dozens of concerned Africans publicly declaring that they will not bow down and become human guinea pigs for the likes of Bill Gates, who is desperately trying to launch new vaccines for the Wuhan coronavirus (COVID-19).

During this protest, some were seen burning their face masks as a symbol of resistance to mass vaccination, which is where things are headed once the human trials are complete, followed by the masks being retired.

Resistance to “colonial medicine,” also known as the pharma injections constantly being imported into Africa by wealthy globalists, is growing to such a degree that even the Bill and Melinda Gates-backed Global Alliance for Vaccines and Immunizations (Gavi) is worried about the future of its eugenics programs.

“In general, people in Africa know the diseases and want to protect each other,” contends Seth Berkley, the white-skinned CEO of Gavi who is concerned that an increasing number of Africans are rejecting his organization’s vaccination efforts.

“In this case, the rumor mill has been dramatic,” he adds, noting that resistance to vaccines in Africa is “the worst [he has] ever seen.”

African Americans all throughout our country’s history have also been targeted with experimental vaccination campaigns. Be sure to check out The Health Ranger Report with Mike Adams, the Health Ranger, to learn more:

Black lives do not matter to Bill Gates or Gavi

In an attempt to justify these vaccine experiments on vulnerable black populations living in Africa, professor of vaccinology Shabir Madhi says that it is an important part of verifying that vaccines work “in the local context,” to quote Fox 5 in New York.

But protesters say that those who are agreeing to participate in such trials do not know the risks involved, and often naively believe that these drug-pushing outsiders are only trying to help them.

“The people chosen as volunteers for the vaccination, they look as if they’re from poor backgrounds, not qualified enough to understand,” says Phapano Phasha, one of the organizers of the protest. “We believe they are manipulating the vulnerable,” he says of Gavi, Gates, and other vaccine pushers.

French researcher Jean-Paul Mira basically admitted to this earlier in the year when he stated that Africa is a perfect place to test experimental vaccines for the Wuhan coronavirus (COVID-19) because “there are no masks, no treatments, no resuscitation.”

“In prostitutes, we try things because we know that they are highly exposed and that they do not protect themselves,” he added, comparing poor, vulnerable blacks living in Africa to prostitutes.

It is this total disregard by wealthy globalists for human lives other than their own that predicates efforts like Gavi’s to test experimental vaccines on the poorest and most trusting populations on earth, many of which live in Africa.

“The narrative we got is our continent is a dumping ground,” laments Phasha, who says that vaccines coming from elsewhere should be tested where they originated before coming to Africa.

“I believe in science,” she is further quoted as saying, emphasizing that vaccine trials should be targeted not just at poor people but at those in all levels of the socioeconomic strata. “And I believe that science has managed to solve most of the problems society is faced with. I’m not against vaccinations; I’m against profiteering.” (Click to Source)

For more related news stories about the dangers of vaccination, be sure to check out Vaccines.news.

Sources for this article include:

Fox5NY.com

NaturalNews.com

Gavi.org

 

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South Africans protest against coronavirus vaccine trials that exploit black lives – but will leftists care?

Sunday, July 05, 2020 by: Ethan Huff

(Natural News) The first human trials for a potential Wuhan coronavirus (COVID-19) vaccine are set to begin in Africa, and protesters in Johannesburg have taken to the streets in protest to declare that they will not be human guinea pigs for this latest Bill Gates depopulation scheme.

Roughly 50 people, mostly black, held up protest signs outside the University of the Witwatersrand (UW) where South Africa’s first clinical trials on a new Wuhan coronavirus (COVID-19) vaccine candidate are set to begin with the help of about 2,000 volunteers.

In partnership with the University of Oxford, UW claims that testing this new vaccine candidate on willing participants will help to ensure that a safe and inexpensive commercial vaccine is eventually made available to Africans. However, the protesters are not so sure.

“I’m not happy at all!” 29-year-old graphic designer Tebogo Legoale told news reporters, reflecting on his continent’s past involvement in testing potentially dangerous drugs on innocent people who do not fully understand the risks involved.

“I mean, this feels like the 1980s all over again when the AIDS pandemic just broke out in South Africa.”

Walter Mashilo, another 29-year-old who is known as a local activist, agrees. He says that he believes members of parliament and ministers’ children should be the first to receive this experimental vaccine, not poor people who are being tricked into potentially sacrificing their own lives.

“We are clear, comrades: we don’t want this vaccine,” Mashilo announced to the crowd standing before him, many of whom were seen carrying “We are not guinea pigs” signage.

The following episode of The Health Ranger Report, which you will not want to miss, discusses the prospect of door-to-door forced vaccinations once one of these experimental Wuhan coronavirus (COVID-19) vaccines is granted market approval:

Traditional healers want their medicines recognized as effective treatment, not new vaccines

With the highest reported infection rate in Africa, South Africa is ground zero for the potential launch of a new Wuhan coronavirus (COVID-19) vaccine, hence why at least one is being tested there. But even traditional healers are saying not so fast.

As is often the case, traditional healing protocols are once again being overlooked or ignored by government officials in Africa, even though they have been used for centuries to help people heal naturally from disease.

Many traditional healers are vocally opposed to the testing of this new vaccine for the Wuhan coronavirus (COVID-19) because they say their methods already work, and do not require the injection of potentially deadly chemicals into people’s bodies.

“We are not going to follow a vaccine because we as healers believe that our traditional medicine is not given a chance,” stated 32-year-old Sellwane Mokatsi, a compliance officer in South Africa who is also a member of the traditional healers’ organization.

Whether one agrees or disagrees with the prospect of using folk medicine to overcome the Wuhan coronavirus (COVID-19), it remains an undeniable fact that innocent black lives are being exploited to rush a new vaccine to market. And the question remains: Do liberals even care?

“If black lives matter – and they do – someone had better straighten these people out,” wrote one commenter at The Globe and Mail who claims to have guest lectured at UW “during the end of Apartheid” when the school was “a beacon of racial tolerance and science-based decision-making.”

“I had great hope for Cyril but he could be presiding over the last democracy,” this same commenter added about South Africa’s current president, Cyril Ramaphosa. “What a sad commentary on South Africa and Wits University.” (Click to Source)

More related news about the Wuhan coronavirus (COVID-19) is available at Pandemic.news.

Sources for this article include:

TheGlobeAndMail.com

NaturalNews.com

 

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DEPOP TAG-TEAM: Anthony Fauci joins Bill Gates in calling for “digital certificates” of coronavirus immunity

Wednesday, April 15, 2020 by: Ethan Huff

(Natural News) In lockstep with Mr. Microsoft (Bill Gates) himself, National Institute of Allergy and Infectious Diseases (NIAID) head Dr. Anthony Fauci has signaled that Americans will eventually be able to return back to normal after the Wuhan coronavirus (COVID-19) crisis ends. But this will probably only be the case if they agree to get vaccinated and carry around digital proof of vaccination everywhere they go.

Speaking on CNN‘s “New Day” program, Fauci stated that requiring digital vaccine certificates is a “possible” outcome of the pandemic because “it’s one of those things that we talk about when we want to make sure that we know who the vulnerable people are and not.”

“This is something that’s being discussed,” he added. “I think it might actually have some merit.”

Louisiana Senator Bill Cassidy, a Republican, has similarly proposed that the government create some type of immune registry to keep track of people who are no longer believed to be at risk of infection with the Wuhan coronavirus (COVID-19). One possible way to do this is to administer antibody testing, which could potentially indicate whether or not someone has already had the Wuhan coronavirus (COVID-19) and is now recovered and immune to it.

Very soon, Fauci says, there will be a “rather large number” of tests available for the Wuhan coronavirus (COVID-19). But a potential problem will be validating them as accurate, seeing as how early Wuhan coronavirus (COVID-19) tests were wildly inaccurate.

“It’s very likely that there are a large number of people out there that have been infected, have been asymptomatic, and did not know they were infected,” Fauci admitted. “If their antibody test is positive, one can formulate kind of strategies about whether or not they would be at risk or vulnerable to get infected.”

Bill Gates and Anthony Fauci want to force the mark of the beast on you in the name of stopping coronavirus

Fauci’s buddy Gates has a similar plan in mind that involves giving people a digital mark of the beast that “approves” them for reentry back into society once they’ve been deemed “safe” following mandatory vaccination for the Wuhan coronavirus (COVID-19).

Gates has stated that he would like to see everyone have a “quantum dot tattoo” microchip inserted into their bodies that would not only “clear” them of the Wuhan coronavirus (COVID-19), but also function as a digital form of identification (ID2020) that includes the entirety of a person’s medical records.

Even though simple zinc is already proving to be a viable “cure” for the Wuhan coronavirus (COVID-19) – no vaccines required – the world’s most outspoken eugenicist at the current time is insistent upon vaccinating the entire globe against this virus in order to keep everyone “safe” from any diseases it might cause.

“The corporate elite that runs things behind the scenes has turned an illness no more dangerous than the common flu into a pretext for an enormous expansion of government power and an enormous transfer of money from the citizens to the corporate elite,” wrote one Washington Times commenter in response to this news.

“Most people still believe – despite so much evidence to the contrary – that the government and the corporate media do not lie, especially about the big things. In reality, the government and the corporate media lie about everything – especially about the big things.” (Click to Source)

To keep up with the latest news about the Wuhan coronavirus (COVID-19), be sure to check out Pandemic.news.

Sources for this article include:

WashingtonTimes.com

NaturalNews.com

NaturalNews.com

 

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New evidence emerges: Coronavirus “bioweapon” might have been a Chinese vaccine experiment gone wrong… genes contain “pShuttle-SN” sequences, proving laboratory origin

Sunday, February 02, 2020 by: Mike Adams

(Natural News) Two days ago, a paper published in the Biorxiv.org journal presented findings that indicated the coronavirus appeared to be engineered with “key structural proteins” of HIV. The paper, entitled, “Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120
and Gag,” concluded that the engineering of coronavirus with such gene sequences was “unlikely to be fortuitous in nature,” providing strong scientific support for the theory that the coronavirus is an engineered bioweapon that escaped laboratory containment in China.

The coverage of this paper by Zero Hedge led to a firestorm of denials by governments, health authorities and the CIA-controlled media, not surprisingly. Any suggestion that the coronavirus was engineered as a bioweapon had to be immediately eliminated. The prevailing panic by the establishment sought to blame this outbreak on Mother Nature — i.e. bats, snakes, seafood, etc. — rather than the human beings who are playing around with deadly biological weapons that are designed to extinguish human life.

Within hours, Twitter slapped down a permanent ban on Zero Hedge, making sure the independent publisher could no longer reach its Twitter audience. After all, the first casualty in any pandemic is the truth, and Jack Dorsey is not only an enabler of pedophiles and child rapists, he’s also an authoritarian tyrant who wants to make sure the public is completely isolated from any “non official” reports about this pandemic. (Jack Dorsey has sided with communist China, in other words. Is anyone surprised?)

Under the intense pressure, the authors of the original paper have now withdrawn the paper and intend to revise it. The publication that originally carried the paper now has a warning message stating, “This article has been withdrawn. Click here for details.” (See original source link here.)

In addition, the publisher has posted a message warning that all the science papers it posts as “preliminary reports” that “should not be regarded as conclusive,” especially when the science establishment is panicked that its official narratives might be crumbling by the hour.

No doubt the authors of this particular paper have been sufficiently threatened to revise their conclusions, and an update of their original paper will soon be posted that effectively denounces everything they stated in the original paper. The criminal wing of the science establishment strikes again, of course, and this tactic of threatening scientists with loss of funding, being blacklisted or even physically threatened and killed is not unusual at all. The CDC, NIH and even the EPA have long histories of threatening scientists with being harmed or killed if they don’t fall in line with the prevailing lies of the establishment. In some cases, they’ve even imprisoned scientists for fraud after those individuals refused to retract their papers. This has been especially common in research areas such as HIV / AIDS, pandemics and vaccines. Any scientist who finds fault with the establishment is destroyed, imprisoned or murdered.

In fact, I’ve interviewed one of the science victims of this, named Judy Mikovits, PhD. Watch my full interview here, but keep reading below first, because there’s a lot more to this story that will shock you:

pShuttle-SN sequence proves the coronavirus pandemic was definitely engineered in a lab

Now, stunning new evidence has emerged that proves the coronavirus was definitely engineered in a laboratory and may have been deliberately injected into patients as part of a Chinese vaccine experiment gone wrong.

As detailed by James Lyons-Weiler, PhD, founder of the Institute for Pure and Applied Knowledge and author of 57 peer-reviewed publications, an analysis of the gene sequence for the coronavirus finds a peculiar sequence called “pShuttle-SN.” This sequence is is the remnant of a genetic engineering sequence that’s used to insert genes into viruses and bacteria. It provides irrefutable “open source” proof that the coronavirus now circulating in the wild was engineered in a laboratory. Every lab that has the gene sequence can see this for themselves. It’s right out in the open, which is why we describe this revelation as “open source.”

“One thing we can say for certain is that this particular virus has a laboratory origin,” states Lyons-Weiler in a bombshell interview with Del Bigtree of The Highwire (see video below, via Brighteon.com, since the video would be banned everywhere else).

This genomic evidence does not, however, prove whether it was produced as a bioweapon or as a vaccine experiment. It could be either one, according to Lyons-Weiler:

In fact, if you then take that sequence and compare it to other proteins, we find that it’s actually a SARS protein that was put into a coronavirus for the purpose of making the vaccine work better. That’s why this element is in there, to create a more reactogenic vaccine.

There’s bombshell after bombshell in this interview, especially at the 27:59 and 33:57 marks. Watch it exclusively at Brighteon.com, given that this information is of course being banned everywhere else by the anti-human tech giants, all of which are now siding with communist China to cover up the truth:

Brighteon.com/105ddb05-07ab-4ef8-8e08-e029dbe5e75c

 

If it was a vaccine experiment gone wrong, then the world is in real trouble, warns analyst

Why does it matter whether the origin was a Chinese vaccine experiment vs. a bioweapon? As previous research has revealed, when these SARS insertions into the coronavirus are introduced into animal as part of a vaccine, they create heightened fatalities when patients are exposed to other coronavirus strains.

In effect, being vaccinated with this particular strain of coronavirus causes individuals to be more easily killed by the common cold and other non-pandemic coronavirus strains that are circulating in the wild.

This is confirmed in the following science paper published in the PLoS ONE science journal: Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus.

This horrifying realization emerged when Chinese researchers were trying to make a SARS vaccine following the SARS outbreak from several years ago. They discovered that the SARS vaccine increased fatalities instead of saving people. Further study revealed that the SARS sequence being inserted into the coronavirus made the virus far more deadly, effectively transforming it into a bioweapon whether intended or not.

Now, James Lyons-Weiler, PhD, has published a detailed article explaining the SARS insertion into the coronavirus and why this is proof that the coronavirus now circulating in the wild is of laboratory origin, not coming from bats and snakes. From his interview, above:

Was it a vaccine that the Chinese government was experimenting with? … If you use a SARS vaccine in ferrets, rats or monkeys, and then they get a secondary infection of a SARS virus, the elderly and immunocompromised end up dying.

So what we may in fact be looking at is a hidden attempt by the Chinese to get a jump on this, the economics of vaccines… it’s not working no matter who develops the vaccine, and that’s why the last time there was a SARS vaccine, the vaccine developers could not get funding because the mice, the rats were dying.

I can say with certainty it’s not just a wild type coronavirus that just happened to acquire this [SARS] element. It’s laboratory acquired somehow. It has escaped from the laboratory or people have been vaccinated with it and that’s why it’s in their bodies…

There’s a huge difference in the predicted health outcome for the world if this is a vaccination experiment in China versus just a laboratory escaped vaccine. In the vaccination experiment, you’re not vaccinated against coronavirus with that vaccine…, so if the Chinese tried to get a jump on the vaccine market by surreptitiously running clinical trials against the coronavirus, and made their elderly and immunocompromised more susceptible to death due to subsequent infection by any coronavirus with this protein that’s in the vaccine, then the rest of the world has a serious humanitarian issue…

…if it is a biological laboratory weapons escape, it’s going to be a nightmare, but if it’s a vaccine type that escaped because of an accidental injection into someone, it’s going to be worse than… if they made their entire population susceptible to SARS with a vaccine program.

In other words, if it’s an escaped vaccine strain that has now gone wild, the entire world is now vulnerable to the SARS-coronavirus gene insertion which was previously found to be killing lab animals, which is why the SARS vaccine program was halted (because it was too dangerous to test on humans).

As Lyons-Weiler writes, “IPAK researchers found a sequence similarity between a pShuttle-SN recombination vector sequence and INS1378. Here’s a shot of the alignment and the DOT Plot.”

This sequence has a 92% match with the Spike protein from the SARS coronavirus:

The process for achieving this was patented by Chinese researchers as shown in this patent link.

Lyons-Weiler warns:

The disease progression in of 2019-nCoV is consistent with those seen in animals and humans vaccinated against SARS and then challenged with re-infection. Thus, the hypothesis that 2019-nCoV is an experimental vaccine type must be seriously considered.

He further concludes that this appears almost certain to be a vaccine experiment gone wrong:

The available evidence most strongly supports that the 2019-NCoV virus is a vaccine strain of coronavirus either accidentally released from a laboratory accident, perhaps a laboratory researcher becoming infected with the virus while conducting animal experiments, or the Chinese were performing clinical studies of a Coronavirus vaccine in humans.

If this is further confirmed, we may, in fact, be looking at a true vaccine holocaust where vaccine researchers are now subjecting the entire world to a deadly mistake that could theoretically cause a very large number of fatalities… all in the quest for vaccine profits, of course.

As Lyons-Weiler further warns:

If the Chinese government has been conducting human trials against SARS. MERS, or other coronviruses using recombined viruses, they may have made their citizens far more susceptible to acute respiratory distress syndrome upon infection with 2019-nCoV coronavirus.

The implications are clear: if China sensitized their population via a SARS vaccine, and this escaped from a lab, the rest of world has a serious humanitarian urgency to help China, but may not expect as serious an epidemic as might otherwise be expected.

In the worst-case scenario, if the vaccination strain is more highly contagious and lethal, 2019-nCoV could become the worst example of vaccine-derived contagious disease in human history.

Tech giants now working over time to cover it all up, censor all dissenting views, and reinforce communist China’s official lies

As expected, the evil tech giants are working overtime to squelch any dissenting views and protect the vaccine industry as well as the bioweapons industry. No blame can be allowed to fall on either one. Instead, Mother Nature must be blamed for all this. And that means the truth must be silenced… a strategy that is, of course, routine for the vaccine industry.

Every channel, voice or website now exploring any human origins of this coronavirus strain — whether related to vaccines or bioweapons — is being rapidly de-platformed and attacked. In some cases, websites are being sabotaged and taken offline, as happened with Natural News last week (we have since recovered, which is why you are able to read this).

Yet the anti-human tech giants are unified in their efforts to silence all information that contradicts official lies, no matter how scientifically accurate that information may be.

Watch this important video to learn more about Big Tech and how it ran a simulation — complete with fake news censorship highlights — to play out the exact scenario we’re all witnessing now: A global pandemic of human origin, infecting tens of thousands of people, accompanied by a government cover-up:

Brighteon.com/b0b9cd83-d97e-4681-a3c5-71f2c0a6607a

(Click to Source)

 

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It’s NOT a Conspiracy Theory! Here’s the PROOF! The Coronavirus was patented here in the US in 2015

I, as well as all of you, have been reading a great deal about the Coronavirus, as it has been rapidly spreading in China and now in other countries (including the US). There have been conspiracy theorists who claim it is a weaponized virus while others bash the idea by coming against that.

So, to remain impartial, I have posted the patent on the Coronavirus for you to make the decision for yourself. Hint: It’s about including it in vaccines as well as using it as a vaccine to prevent(???) an array of diseases. Decide for yourselves. In the end, we all have to formulate what we think is the truth and placing this information here is by no means my way of persuading you one way or another.

Please Note: The ‘Tables’ on the original patent information would not cooperate with me to copy and past them in their appropriate places, so if you’re curious, just click to the patent itself.

The Coronavirus’ US patent number is: 10130701

The patent information below is found by CLICKING HERE.

Coronavirus

Jul 23, 2015 – THE PIRBRIGHT INSTITUTE

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.

Latest THE PIRBRIGHT INSTITUTE Patents:

Skip to: Description  ·  Claims  ·  References Cited  · Patent History  ·  Patent HistoryDescriptionFIELD 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 SEQ ID NO: 1 ACTTAAGATAGATATTAATATATATCTATCACACTAGCCTTGCGCTAGATTTCCAACTTA ACAAAACGGACTTAAATACCTACAGCTGGTCCTCATAGGTGTTCCATTGCAGTGCACTTT AGTGCCCTGGATGGCACCTGGCCACCTGTCAGGTTTTTGTTATTAAAATCTTATTGTTGC TGGTATCACTGCTTGTTTTGCCGTGTCTCACTTTATACATCCGTTGCTTGGGCTACCTAG TATCCAGCGTCCTACGGGCGCCGTGGCTGGTTCGAGTGCGAAGAACCTCTGGTTCATCTA GCGGTAGGCGGGTGTGTGGAAGTAGCACTTCAGACGTACCGGTTCTGTTGTGTGAAATAC GGGGTCACCTCCCCCCACATACCTCTAAGGGCTTTTGAGCCTAGCGTTGGGCTACGTTCT CGCATAAGGTCGGCTATACGACGTTTGTAGGGGGTAGTGCCAAACAACCCCTGAGGTGAC AGGTTCTGGTGGTGTTTAGTGAGCAGACATACAATAGACAGTGACAACATGGCTTCAAGC CTAAAACAGGGAGTATCTGCGAAACTAAGGGATGTCATTGTTGTATCCAAAGAGATTGCT GAACAACTTTGTGACGCTTTGTTTTTCTATACGTCACACAACCCTAAGGATTACGCTGAT GCTTTTGCAGTTAGGCAGAAGTTTGATCGTAATCTGCAGACTGGGAAACAGTTCAAATTT GAAACTGTGTGTGGTCTCTTCCTCTTGAAGGGAGTTGACAAAATAACACCTGGCGTCCCA GCAAAAGTCTTAAAAGCCACTTCTAAGTTGGCAGATTTAGAAGACATCTTTGGTGTCTCT CCCTTTGCAAGAAAATATCGTGAACTTTTGAAGACAGCATGCCAGTGGTCTCTTACTGTA GAAACACTGGATGCTCGTGCACAAACTCTTGATGAAATTTTTGACCCTACTGAAATACTT TGGCTTCAGGTGGCAGCAAAAATCCAAGTTTCGGCTATGGCGATGCGCAGGCTTGTTGGA GAAGTAACTGCAAAAGTCATGGATGCTTTGGGCTCAAATATGAGTGCTCTTTTCCAGATT TTTAAACAACAAATAGTCAGAATTTTTCAAAAAGCGCTGGCTATTTTTGAGAATGTGAGT GAATTACCACAGCGTATTGCAGCACTTAAGATGGCTTTTGCTAAGTGTGCCAAGTCCATT ACTGTTGTGGTTATGGAGAGGACTCTAGTTGTTAGAGAGTTCGCAGGAACTTGTCTTGCA AGCATTAATGGTGCTGTTGCAAAATTCTTTGAAGAACTCCCAAATGGTTTCATGGGTGCT AAAATTTTCACTACACTTGCCTTCTTTAGGGAGGCTGCAGTGAAAATTGTGGATAACATA CCAAATGCACCGAGAGGCACTAAAGGGTTTGAAGTCGTTGGTAATGCCAAAGGTACACAA GTTGTTGTGCGTGGCATGGGAAATGACTTAACACTGGTTGAGCAAAAAGCTGAAATTGCT GTGGAGTCAGAAGGTTGGTCTGCAATTTTGGGTGGACATCTTTGCTATGTCTTTAAGAGT GGTGATCGCTTTTACGCGGCACCTCTTTCAGGAAATTTTGCATTGCATGATGTGCATTGT TGTGAGCGTGTTGTCTGTCTTTCTGATGGTGTAACACCGGAGATAAATGATGGACTTATT CTTGCAGCAATCTACTCTTCTTTTAGTGTCGCAGAACTTGTGGCAGCCATTAAAAGGGGT GAACCATTTAAGTTTCTGGGTCATAAATTTGTGTATGCAAAGGATGCAGCAGTTTCTTTT ACATTAGCGAAGGCTGCTACTATTGCAGATGTTTTGAAGCTGTTTCAATCAGCGCGTGTG AAAGTAGAAGATGTTTGGTCTTCACTTACTGAAAAGTCTTTTGAATTCTGGAGGCTTGCA TATGGAAAAGTGCGTAATCTCGAAGAATTTGTTAAGACTTGTTTTTGTAAGGCTCAAATG GCGATTGTGATTTTAGCGACAGTGCTTGGAGAGGGCATTTGGCATCTTGTTTCGCAAGTC ATCTATAAAGTAGGTGGTCTTTTTACTAAAGTTGTTGACTTTTGTGAAAAATATTGGAAA GGTTTTTGTGCACAGTTGAAAAGAGCTAAGCTCATTGTCACTGAAACCCTCTGTGTTTTG AAAGGAGTTGCACAGCATTGTTTTCAACTATTGCTGGATGCAATACAGTTTATGTATAAA AGTTTTAAGAAGTGTGCACTTGGTAGAATCCATGGAGACTTGCTCTTCTGGAAAGGAGGT GTGCACAAAATTATTCAAGAGGGCGATGAAATTTGGTTTGAGGGCATTGATAGTATTGAT GTTGAAGATCTGGGTGTTGTTCAAGAAAAATTGATTGATTTTGATGTTTGTGATAATGTG ACACTTCCAGAGAACCAACCCGGTCATATGGTTCAAATCGAGGATGACGGAAAGAACTAC ATGTTCTTCCGCTTCAAAAAGGATGAGAACATTTATTATACACCAATGTCACAGCTTGGT GCTATTAATGTGGTTTGCAAAGCAGGCGGTAAAACTGTCACCTTTGGAGAAACTACTGTG CAAGAAATACCACCACCTGATGTTGTGTTTATTAAGGTTAGCATTGAGTGTTGTGGTGAA CCATGGAATACAATCTTCAAAAAGGCTTATAAGGAGCCCATTGAAGTAGAGACAGACCTC ACAGTTGAACAATTGCTCTCTGTGGTCTATGAGAAAATGTGTGATGATCTCAAGCTGTTT CCGGAGGCTCCAGAACCACCACCATTTGAGAATGTCACACTTGTTGATAAGAATGGTAAA GATTTGGATTGCATAAAATCATGCCATCTGATCTATCGTGATTATGAGAGCGATGATGAC ATCGAGGAAGAAGATGCAGAAGAATGTGACACGGATTCAGGTGATGCTGAGGAGTGTGAC ACTAATTCAGAATGTGAAGAAGAAGATGAGGATACTAAAGTGTTGGCTCTTATACAAGAC CCGGCAAGTAACAAATATCCTCTGCCTCTTGATGATGATTATAGCGTCTACAATGGATGT ATTGTTCATAAGGACGCTCTCGATGTTGTGAATTTACCATCTGGTGAAGAAACCTTTGTT GTCAATAACTGCTTTGAAGGGGCTGTTAAAGCTCTTCCGCAGAAAGTTATTGATGTTCTA GGTGACTGGGGTGAGGCTGTTGATGCGCAAGAACAATTGTGTCAACAAGAATCAACTCGG GTCATATCTGAGAAATCAGTTGAGGGTTTTACTGGTAGTTGTGATGCAATGGCTGAACAA GCTATTGTTGAAGAGCAGGAAATAGTACCTGTTGTTGAACAAAGTCAGGATGTAGTTGTT TTTACACCTGCAGACCTAGAAGTTGTTAAAGAAACAGCAGAAGAGGTTGATGAGTTTATT CTCATTTCTGCTGTCCCTAAAGAAGAAGTTGTGTCTCAGGAGAAAGAGGAGCCACAGGTT GAGCAAGAGCCTACCCTAGTTGTTAAAGCACAACGTGAGAAGAAGGCTAAAAAGTTCAAA GTTAAACCAGCTACATGTGAAAAACCCAAATTTTTGGAGTACAAAACATGTGTGGGTGAT TTGGCTGTTGTAATTGCCAAAGCATTGGATGAGTTTAAAGAGTTCTGCATTGTAAACGCT GCAAATGAGCACATGTCGCATGGTGGTGGCGTTGCAAAGGCAATTGCAGACTTTTGTGGA CCGGACTTTGTTGAATATTGCGCGGACTATGTTAAGAAACATGGTCCACAGCAAAAACTT GTCACACCTTCATTTGTTAAAGGCATTCAATGTGTGAATAATGTTGTAGGACCTCGCCAT GGAGACAGCAACTTGCGTGAGAAGCTTGTTGCTGCTTACAAGAGTGTTCTTGTAGGTGGA GTGGTTAACTATGTTGTGCCAGTTCTCTCATCAGGGATTTTTGGTGTAGATTTTAAAATA TCAATAGATGCTATGCGCGAAGCTTTTAAAGGTTGTGCCATACGCGTTCTTTTATTTTCT CTGAGTCAAGAACACATCGATTATTTCGATGCAACTTGTAAGCAGAAGACAATTTATCTT ACGGAGGATGGTGTTAAATACCGCTCTGTTGTTTTAAAACCTGGTGATTCTTTGGGTCAA TTTGGACAGGTTTTTGCAAGAAATAAGGTAGTCTTTTCGGCTGATGATGTTGAGGATAAA GAAATCCTCTTTATACCCACAACTGACAAGACTATTCTTGAATATTATGGTTTAGATGCG CAAAAGTATGTAACATATTTGCAAACGCTTGCGCAGARATGGGATGTTCAATATAGAGAC AATTTTGTTATATTAGAGTGGCGTGACGGAAATTGCTGGATTAGTTCAGCAATAGTTCTC CTTCAAGCTGCTAAAATTAGATTTAAAGGTTTTCTTGCAGAAGCATGGGCTAAACTGTTG GGTGGAGATCCTACAGACTTTGTTGCCTGGTGTTATGCAAGTTGCAATGCTAAAGTAGGT GATTTTTCAGATGCTAATTGGCTTTTGGCCAATTTAGCAGAACATTTTGACGCAGATTAC ACAAATGCACTTCTTAAGAAGTGTGTGTCGTGCAATTGTGGTGTTAAGAGTTATGAACTT AGGGGTCTTGAAGCCTGTATTCAGCCAGTTCGAGCACCTAATCTTCTACATTTTAAAACG CAATATTCAAATTGCCCAACCTGTGGTGCAAGTAGTACGGATGAAGTAATAGAAGCTTCA TTACCGTACTTATTGCTTTTTGCTACTGATGGTCCTGCTACAGTTGATTGTGATGAAAAT GCTGTAGGGACTGTTGTTTTCATTGGCTCTACTAATAGTGGCCATTGTTATACACAAGCC GATGGTAAGGCTTTTGACAATCTTGCTAAGGATAGAAAATTTGGAAGGAAGTCGCCTTAC ATTACAGCAATGTATACACGTTTTTCTCTTAGGAGTGAAAATCCCCTACTTGTTGTTGAA CATAGTAAGGGTAAAGCTAAAGTAGTAAAAGAAGATGTTTCTAACCTTGCTACTAGTTCT AAAGCCAGTTTTGACGATCTTACTGACTTTGAACACTGGTATGATAGCAACATCTATGAG AGTCTTAAAGTGCAGGAGACACCTGATAATCTTGATGAATATGTGTCATTTACGACAAAG GAAGATTCTAAGTTGCCACTGACACTTAAAGTTAGAGGTATCAAATCAGTTGTTGACTTT AGGTCTAAGGATGGTTTTACTTATAAGTTAACACCTGATACTGATGAAAATTCAAAAACA CCAGTCTACTACCCAGTCTTGGATTCTATTAGTCTTAGGGCAATATGGGTTGAAGGCAGT GCTAATTTTGTTGTTGGGCATCCAAATTATTATAGTAAGTCTCTCCGAATTCCCACGTTT TGGGAAAATGCCGAGAGCTTTGTTAAAATGGGTTATAAAATTGATGGTGTAACTATGGGC CTTTGGCGTGCAGAACACCTTAATAAACCTAATTTGGAGAGAATTTTTAACATTGCTAAG AAAGCTATTGTTGGATCTAGTGTTGTTACTACGCAGTGTGGTAAAATACTAGTTAAAGCA GCTACATACGTTGCCGATAAAGTAGGTGATGGTGTAGTTCGCAATATTACAGATAGAATT AAGGGTCTTTGTGGATTCACACGTGGCCATTTTGAAAAGAAAATGTCCCTACAATTTCTA AAGACACTTGTGTTCTTTTTCTTTTATTTCTTAAAGGCTAGTGCTAAGAGTTTAGTTTCT AGCTATAAGATTGTGTTATGTAAGGTGGTGTTTGCTACCTTACTTATAGTGTGGTTTATA TACACAAGTAATCCAGTAGTGTTTACTGGAATACGTGTGCTAGACTTCCTATTTGAAGGT TCTTTATGTGGTCCTTATAATGACTACGGTAAAGATTCTTTTGATGTGTTACGGTATTGT GCAGGTGATTTTACTTGTCGTGTGTGTTTACATGATAGAGATTCACTTCATCTGTACAAA CATGCTTATAGCGTAGAACAAATTTATAAGGATGCAGCTTCTGGCATTAACTTTAATTGG AATTGGCTTTATTTGGTCTTTCTAATATTATTTGTTAAGCCAGTGGCAGGTTTTGTTATT ATTTGTTATTGTGTTAAGTATTTGGTATTGAGTTCAACTGTGTTGCAAACTGGTGTAGGT TTTCTAGATTGGTTTGTAAAAACAGTTTTTACCCATTTTAATTTTATGGGAGCGGGATTT TATTTCTGGCTCTTTTACAAGATATACGTACAAGTGCATCATATATTGTACTGTAAGGAT GTAACATGTGAAGTGTGCAAGAGAGTTGCACGCAGCAACAGGCAAGAGGTTAGCGTTGTA GTTGGTGGACGCAAGCAAATAGTGCATGTTTACACTAATTCTGGCTATAACTTTTGTAAG AGACATAATTGGTATTGTAGAAATTGTGATGATTATGGTCACCAAAATACATTTATGTCC CCTGAAGTTGCTGGCGAGCTTTCTGAAAAGCTTAAGCGCCATGTTAAACCTACAGCATAT GCTTACCACGTTGTGTATGAGGCATGCGTGGTTGATGATTTTGTTAATTTAAAATATAAG GCTGCAATTGCTGGTAAGGATAATGCATCTTCTGCTGTTAAGTGTTTCAGTGTTACAGAT TTTTTAAAGAAAGCTGTTTTTCTTAAGGAGGCATTGAAATGTGAACAAATATCTAATGAT GGTTTTATAGTGTGTAATACACAGAGTGCGCATGCACTAGAGGAAGCAAAGAATGCAGCC GTCTATTATGCGCAATATCTGTGTAAGCCAATACTTATACTTGACCAGGCACTTTATGAG CAATTAATAGTAGAGCCTGTGTCTAAGAGTGTTATAGATAAAGTGTGTAGCATTTTGTCT AATATAATATCTGTAGATACTGCAGCTTTAAATTATAAGGCAGGCACACTTCGTGATGCT CTGCTTTCTATTACTAAAGACGAAGAAGCCGTAGATATGGCTATCTTCTGCCACAATCAT GAAGTGGAATACACTGGTGACGGTTTTACTAATGTGATACCGTCATATGGTATGGACACT GATAAGTTGACACCTCGTGATAGAGGGTTTTTGATAAATGCAGATGCTTCTATTGCTAAT TTAAGAGTCAAAAATGCTCCTCCGGTAGTATGGAAGTTTTCTGATCTTATTAAATTGTCT GACAGTTGCCTTAAATATTTAATTTCAGCTACTGTCAAGTCAGGAGGTCGTTTCTTTATA ACAAAGTCTGGTGCTAAACAAGTTATTTCTTGTCATACCCAGAAACTGTTGGTAGAGAAA AAGGCAGGTGGTGTTATTAATAACACTTTTAAATGGTTTATGAGTTGTTTTAAATGGCTT TTTGTCTTTTATATACTTTTTACAGCATGTTGTTTGGGTTACTACTATATGGAGATGAAT AAAAGTTTTGTTCACCCCATGTATGATGTAAACTCCACACTGCATGTTGAAGGGTTCAAA GTTATAGACAAAGGTGTTATTAGAGAGATTGTGTCAGAAGATAATTGTTTCTCTAATAAG TTTGTTAATTTTGACGCCTTTTGGGGTAAATCATATGAAAATAATAAAAACTGTCCAATT GTTACAGTTGTTATAGATGGTGACGGGACAGTAGCTGTTGGTGTTCCTGGTTTTGTATCA TGGGTTATGGATGGTGTTATGTTTGTGCATATGACACAGACTGATCGTAGACCTTGGTAC ATTCCTACCTGGTTTAATAGAGAAATTGTTGGTTACACTCAGGATTCAATTATCACTGAG GGTAGTTTTTATACATCTATAGCATTATTTTCTGCTAGATGTTTATATTTAACAGCCAGC AATACACCTCAATTGTATTGTTTTAATGGCGACAATGATGCACCTGGAGCCTTACCATTT GGTAGTATTATTCCTCATAGAGTATACTTCCAACCTAATGGTGTTAGGCTTATAGTTCCA CAACAAATACTGCATACACCCTACATAGTGAAGTTTGTTTCAGACAGCTATTGTAGAGGT AGTGTATGTGAGTATACTAAACCAGGTTACTGTGTGTCACTAGACTCCCAATGGGTTTTG TTTAATGATGAATACATTAGTAAACCTGGCGTTTTCTGTGGTTCTACTGTTAGAGAACTT ATGTTTAATATGGTTAGTACATTCTTTACTGGTGTCAACCCTAATATTTATATTCAGCTA GCAACTATGTTTTTAATACTAGTTGTTATTGTGTTAATTTTTGCAATGGTTATAAAGTTT CAAGGTGTTTTTAAAGCTTATGCGACCATTGTGTTTACAATAATGTTAGTTTGGGTTATT AATGCATTTGTTTTGTGTGTACATAGTTATAATAGTGTTTTAGCTGTTATATTATTAGTA CTCTATTGCTATGCATCATTGGTTACAAGTCGCAATACTGCTATAATAATGCATTGTTGG CTTGTTTTTACCTTTGGTTTAATAGTACCCACATGGTTGGCTTGTTGCTATCTGGGATTT ATTCTTTATATGTACACACCGTTGGTTTTCTGGTGTTACGGTACTACTAAAAATACTCGT AAGTTGTATGATGGCAACGAGTTTGTTGGTAATTATGACCTTGCTGCGAAGAGCACTTTT GTTATTCGTGGTACTGAATTTGTTAAGCTTACGAATGAGATAGGTGATAAATTTGAAGCC TATCTTTCTGCGTATGCTAGACTTAAATACTATTCAGGCACTGGTAGTGAGCAAGATTAC TTGCAAGCTTGTCGTGCATGGTTAGCTTATGCTTTGGACCAATATAGAAATAGTGGTGTT GAGGTTGTTTATACCCCACCGCGTTACTCTATTGGTGTTAGTAGACTACACGCTGGTTTT AAAAAACTAGTTTCTCCTAGTAGTGCTGTTGAGAAGTGCATTGTTAGTGTCTCTTATAGA GGCAATAATCTTAATGGACTGTGGCTGGGTGATTCTATTTACTGCCCACGCCATGTGTTA GGTAAGTTTAGTGGTGACCAGTGGGGTGACGTACTAAACCTTGCTAATAATCATGAGTTT GAAGTTGTAACTCAAAATGGTGTTACTTTGAATGTTGTCAGCAGGCGGCTTAAAGGAGCA GTTTTAATTTTACAAACTGCAGTTGCCAATGCTGAAACTCCTAAGTATAAGTTTGTTAAA GCTAATTGTGGTGATAGTTTCACTATAGCTTGTTCTTATGGTGGTACAGTTATAGGACTT TACCCTGTCACTATGCGTTCTAATGGTACTATTAGAGCATCTTTCCTAGCAGGAGCCTGT GGCTCAGTTGGTTTTAATATAGAAAAGGGTGTAGTTAATTTCTTTTATATGCACCATCTT GAGTTACCTAATGCATTACACACTGGAACTGACCTAATGGGTGAGTTTTATGGTGGTTAT GTAGATGAAGAGGTTGCGCAAAGAGTGCCACCAGATAATCTAGTTACTAACAATATTGTA GCATGGCTCTATGGGGCAATTATTAGTGTTAAAGAAAGTAGTTTTTCACAACCTAAATGG TTGGAGAGTACTACTGTTTCTATTGAAGATTACAATAGGTGGGCTAGTGATAATGGTTTT ACTCCATTTTCCACTAGTACTGCTATTACTAAATTAAGTGCTATAACTGGGGTTGATGTT TGTAAACTCCTTCGCACTATTATGGTAAAAAGTGCTCAATGGGGTAGTGATCCCATTTTA GGACAATATAATTTTGAAGACGAATTGACACCAGAATCTGTATTTAATCAAGTTGGTGGT GTTAGGTTACAGTCTTCTTTTGTAAGAAAAGCTACATCTTGGTTTTGGAGTAGATGTGTA TTAGCTTGCTTCTTGTTTGTGTTGTGTGCTATTGTCTTATTTACGGCAGTGCCACTTAAG TTTTATGTACATGCAGCTGTTATTTTGTTGATGGCTGTGCTCTTTATTTCTTTTACTGTT AAACATGTTATGGCATACATGGACACTTTCCTATTGCCTACATTGATTACAGTTATTATT GGAGTTTGTGCTGAAGTCCCTTTCATATACAATACTCTAATTAGTCAAGTTGTTATTTTC TTAAGCCAATGGTATGATCCTGTAGTCTTTGATACTATGGTACCATGGATGTTATTGCCA TTAGTGTTGTACACTGCTTTTAAGTGTGTACAAGGCTGCTATATGAATTCTTTCAATACT TCTTTGTTAATGCTGTATCAGTTTATGAAGTTAGGTTTTGTTATTTACACCTCTTGAAAC ACTCTTACTGCATATACAGAAGGTAATTGGGAGTTATTCTTTGAGTTGGTTCACACTATT GTGTTGGCTAATGTTAGTAGTAATTCCTTAATTGGTTTAATTGTTTTTAAGTGTGCTAAG TGGATTTTATATTATTGCAATGCAACATACTTTAATAATTATGTGTTAATGGCAGTCATG GTTAATGGCATAGGCTGGCTTTGCACCTGTTACTTTGGATTGTATTGGTGGGTTAATAAA GTTTTTGGTTTAACCTTAGGTAAATACAATTTTAAAGTTTCAGTAGATCAATATAGGTAT ATGTGTTTGCATAAGGTAAATCCACCTAAAACTGTGTGGGAGGTCTTTACTACAAATATA CTTATACAAGGAATTGGAGGCGATCGTGTGTTGCCTATAGCTACAGTGCAATCTAAATTG AGTGATGTAAAGTGTACAACTGTTGTTTTAATGCAGCTTTTGACTAAGCTTAATGTTGAA GCAAATTCAAAAATGCATGCTTATCTTGTTGAGTTACACAATAAAATCCTCGCATCTGAT GATGTTGGAGAGTGCATGGATAATTTATTGGGTATGCTTATAACACTATTTTGTATAGAT TCTACTATTGATTTGGGTGAGTATTGTGATGATATACTTAAGAGGTCAACTGTATTACAA TCGGTTACTCAAGAGTTTTCGCACATACCCTCGTATGCTGAATATGAAAGAGCTAAGAGT ATTTATGAAAAGGTTTTAGCCGATTCTAAAAATGGTGGTGTAACACAGCAAGAGCTTGCT GCATATCGTAAAGCTGCCAATATTGCAAAGTCAGTTTTTGATAGAGACTTGGCTGTTCAA AAGAAGTTAGATAGCATGGCAGAACGTGCTATGACAACAATGTATAAAGAGGCGCGTGTA ACTGATAGAAGAGCAAAATTAGTTTCATCATTACATGCACTACTTTTTTCAATGCTTAAG AAAATAGATTCTGAGAAGCTTAATGTCTTATTTGACCAGGCGAATAGTGGTGTTGTACCC CTAGCAACTGTTCCAATTGTTTGTAGTAATAAGCTTACCCTTGTTATACCAGACCCAGAG ACGTGGGTCAAGTGTGTGGAGGGTGTGCATGTTACATATTCAACAGTTGTTTGGAATATA GACTGTGTTACTGATGCCGATGGCACAGAGTTACACCCCACTTCTACAGGTAGTGGATTG ACTTACTGTATAAGTGGTGATAATATAGCATGGCCTTTAAAGGTTAACTTGACTAGGAAT GGGCATAATAAGGTTGATGTTGCCTTGCAAAATAATGAGCTTATGCCTCACGGTGTAAAG ACAAAGGCTTGCGTAGCAGGTGTAGATCAAGCACATTGTAGCGTTGAGTCTAAATGTTAT TATACAAGTATTAGTGGCAGTTCAGTTGTAGCTGCTATTACCTCTTCAAATCCTAATCTG AAAGTAGCCTCTTTTTTGAATGAGGCAGGTAATCAGATTTATGTAGACTTAGACCGAGCA TGTAAATTTGGTATGAAAGTGGGTGATAAGGTTGAAGTTGTTTACCTGTATTTTATAAAA AATACGAGGTCTATTGTAAGAGGTATGGTACTTGGTGCTATATCTAATGTTGTTGTGTTA CAATCTAAAGGTCATGAGACAGAGGAAGTGGATGCTGTAGGCATTCTCTCACTTTGTTCT TTTGCAGTAGATCCTGCGGATACATATTGTAAATATGTGGCAGCAGGTAATCAACCTTTA GGTAACTGTGTTAAAATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAG CCAAGTCCAACTCCGGATCAGGATTCTTATGGAGGAGCTTCTGTGTGTCTTTATTGTAGA GCACATATAGCACACCCTGGCGGAGCAGGAAATTTAGATGGACGCTGTCAATTTAAAGGT TCTTTTGTGCAAATACCTACTACGGAGAAAGATCCTGTTGGATTCTGTCTACGTAACAAG GTTTGCACTGTTTGTCAGTGTTGGATTGGTTATGGATGTCAGTGTGATTCACTTAGACAA CCTAAACCTTCTGTTCAGTCAGTTGCTGTTGCATCTGGTTTTGATAAGAATTATTTAAAC GGGTACGGGGTAGCAGTGAGGCTCGGCTGATACCCCTAGCTAATGGATGTGACCCCGATG TTGTAAAGCGAGCCTTTGATGTTTGTAATAAGGAATCAGCCGGTATGTTTCAAAATTTGA AGCGTAACTGTGCACGATTCCAAGAAGTACGTGATACTGAAGATGGAAATCTTGAGTATT GTGATTCTTATTTTGTGGTTAAACAAACCACTCCTAGTAATTATGAACATGAGAAAGCTT GTTATGAAGACTTAAAGTCAGAAGTAACAGCTGATCATGATTTCTTTGTGTTCAATAAGA ACATTTATAATATTAGTAGGCAGAGGCTTACTAAGTATACTATGATGGATTTTTGCTATG CTTTGCGGCACTTTGACCCAAAGGATTGCGAAGTTCTTAAAGAAATACTTGTCACTTATG GTTGTATAGAAGATTATCACCCTAAGTGGTTTGAAGAGAATAAGGATTGGTACGACCCAA TAGAAAACCCTAAATATTATGCCATGTTGGCTAAAATGGGACCTATTGTACGAGGTGCTT TATTGAATGCTATTGAGTTCGGAAACCTCATGGTTGAAAAAGGTTATGTTGGTGTTATTA CACTTGATAACCAAGATCTTAATGGCAAATTTTATGATTTTGGTGATTTTCAGAAGACAG CGCCTGGTGCTGGTGTTCCTGTTTTTGATACGTATTATTCTTACATGATGCCCATCATAG CCATGACTGATGCGTTGGCACCTGAGAGGTATTTTGAATATGATGTGCATAAGGGTTATA AATCTTATGATCTCCTCAAGTATGATTATACTGAGGAGAAACAAGATTTGTTTCAGAAGT ACTTTAAGTATTGGGATCAAGAGTATCACCCTAACTGTCGCGACTGTAGTGATGACAGGT GTTTGATACATTGTGCAAACTTCAACATCTTGTTTTCTACACTTGTACCGCAGACTTCTT TCGGTAATTTGTGTAGAAAGGTTTTTGTTGATGGTGTACCATTTATAGCTACTTGTGGCT ATCATTCTAAGGAACTTGGTGTTATTATGAATCAAGATAACACCATGTCATTTTCAAAAA TGGGTTTGAGTGAACTCATGGAGTTTGTTGGAGATCGTGGCTTGTTAGTGGGGACATGCA ATAAATTAGTGGATCTTAGAACGTCTTGTTTTAGTGTTTGTGCTTTAGCGTCTGGTATTA CTCATCAAACGGTAAAACCAGGTCACTTTAACAAGGATTTCTACGATTTTGCAGAGAAGG CTGGTATGTTTAAGGAAGGTTCTTCTATACCACTTAAACATTTCTTCTACCCACAGACTG GTAATGCTGCTATAAACGATTATGATTATTATCGTTATAACAGGCCTACCATGTTTGATA TACGTCAACTTTTATTTTGTTTAGAAGTGACTTCTAAATATTTTGAATGTTATGAAGGCG GCTGTATACCAGCAAGCCAAGTTGTAGTTAACAATTTAGATAAGAGTGCAGGTTATCCGT TCAATAAGTTTGGAAAGGCCCGTCTCTATTATGAAATGAGTCTAGAGGAGCAGGACCAAC TCTTTGAGAGTACAAAGAAGAACGTCCTGCCTACTATAACTCAGATGAATTTAAAATATG CCATATCCGCGAAAAATAGAGCGCGTACAGTGGCAGGTGTGTCTATCCTTTCTACTATGA CTAATAGGCAGTTTCATCAGAAGATTCTTAAGTCTATAGTCAACACTAGAAACGCTCCTG TAGTTATTGGAACAACCAAGTTTTATGGCGGTTGGGATAACATGTTGAGAAACCTTATTC AGGGTGTTGAAGACCCGATTCTTATGGGTTGGGATTATCCAAAGTGTGATAGAGCAATGC CTAATTTGTTGCGTATAGCAGCATCTTTAGTACTCGCTCGTAAACACACTAATTGTTGTA CTTGGTCTGAACGCGTTTATAGGTTGTATAATGAATGCGCTCAGGTTTTATCTGAAACTG TCTTAGCTACAGGTGGTATATATGTGAAACCTGGTGGTACTAGCAGTGGAGATGCTACTA CTGCTTATGCAAACAGTGTTTTCAACATAATACAAGCCACATCTGCTAATGTTGCGCGTC TTTTGAGTGTTATAACGCGTGATATTGTATATGATGACATTAAGAGCTTGCAGTATGAAT TGTACCAGCAGGTTTATAGGCGAGTCAATTTTGACCCAGCATTTGTTGAAAAGTTTTATT CTTATTTGTGTAAGAATTTCTCATTGATGATCTTGTCTGACGACGGTGTTGTTTGTTATA ACAACACATTAGCCAAACAAGGTCTTGTAGCAGATATTTCTGGTTTTAGAGAAGTTCTCT ACTATCAGAACAATGTTTTTATGGCTGATTCTAAATGTTGGGTTGAACCAGATTTAGAAA AAGGCCCACATGAATTTTGTTCACAGCACACAATGTTAGTGGAGGTTGATGGTGAGCCTA GATACTTGCCATATCCAGACCCATCACGTATTTTGTGTGCATGTGTTTTTGTAGATGATT TGGATAAGACAGAATCTGTGGCTGTTATGGAGCGTTATATCGCTCTTGCCATAGATGCGT ACCCACTAGTACATCATGAAAATGAGGAGTACAAGAAGGTATTCTTTGTGCTTCTTTCAT ACATCAGAAAACTCTATCAAGAGCTTTCTCAGAATATGCTTATGGACTACTCTTTTGTAA TGGATATAGATAAGGGTAGTAAATTTTGGGAACAGGAGTTCTATGAAAATATGTATAGAG CCCCTACAACATTACAGTGTTGTGGCGTTTGTGTAGTGTGTAATAGTCAAACTATATTGC GCTGTGGTAATTGTATTCGCAAACCATTTTTGTGTTGTAAGTGTTGCTATGACCATGTCA TGCACACAGACCACAAAAATGTTTTGTCTATAAATCCTTACATTTGCTCACAGCCAGGTT GTGGTGAAGCAGATGTTACTAAATTGTACCTCGGAGGTATGTCATACTTCTGCGGTAATC ATAAACCAAAGTTATCAATACCGTTAGTATCTAATGGTACAGTGTTTGGAATTTACAGGG CTAATTGTGCAGGTAGCGAAAATGTTGATGATTTTAATCAACTAGCTACTACTAATTGGT CTACTGTGGAACCTTATATTTTGGCAAATCGTTGTGTAGATTCGTTGAGACGCTTTGCTG CAGAGACAGTAAAAGCTACAGAAGAATTACATAAGCAACAATTTGCTAGTGCAGAAGTGA GAGAAGTACTCTCAGATCGTGAATTGATTCTGTCTTGGGAGCCAGGTAAAACCAGGCCTC CATTGAATAGAAATTATGTTTTCACTGGCTTTCACTTTACTAGAACTAGTAAAGTTCAGC TCGGTGATTTTACATTTGAAAAAGGTGAAGGTAAGGACGTTGTCTATTATCGAGCGACGT CTACTGCTAAATTGTCTGTTGGAGACATTTTTGTTTTAACCTCACACAATGTTGTTTCTC TTATAGCGCCAACGTTGTGTCCTCAGCAAACCTTTTCTAGGTTTGTGAATTTAAGACCTA ATGTGATGGTACCTGCGTGTTTTGTAAATAACATTCCATTGTACCATTTAGTAGGCAAGC AGAAGCGTACTACAGTACAAGGCCCTCCTGGCAGTGGTAAATCCCATTTTGCTATAGGAT TGGCGGCTTACTTTAGTAACGCCCGTGTCGTTTTTACTGCATGCTCTCATGCAGCTGTTG ATGCTTTATGTGAAAAAGCTTTTAAGTTTCTTAAAGTAGATGATTGCACTCGTATAGTAC CTCAAAGGACTACTATCGATTGCTTCTCTAAGTTTAAAGGTAATGACACAGGCAAAAAGT ACATTTTTAGTACTATTAATGCCTTGCCAGAAGTTAGTTGTGACATTCTTTTGGTTGACG AGGTTAGTATGTTGACCAATTACGAATTGTCTTTTATTAATGGTAAGATAAACTATCAAT ATGTTGTGTATGTAGGTGATCCTGCTCAATTACCGGCGCCTCGTACGTTGCTTAACGGTT CACTCTCTCCAAAGGATTATAATGTTGTCACAAACCTTATGGTTTGTGTTAAACCTGACA TTTTCCTTGCAAAGTGTTACCGTTGTCCTAAAGAAATTGTAGATACTGTTTCTACTCTTG TATATGATGGAAAGTTTATTGCAAATAACCCGGAATCACGTCAGTGTTTCAAGGTTATAG TTAATAATGGTAATTCTGATGTAGGACATGAAAGTGGCTCAGCCTACAACATAACTCAAT TAGAATTTGTGAAAGATTTTGTCTGTCGCAATAAGGAATGGCGGGAAGCAACATTCATTT CACCTTATAATGCTATGAACCAGAGAGCCTACCGTATGCTTGGACTTAATGTTCAGACAG TAGACTCGTCTCAAGGTTCGGAGTATGATTATGTTATCTTTTGTGTTACTGCAGATTCGC AGCATGCACTGAATATTAACAGATTCAATGTAGCGCTTACAAGAGCCAAGCGTGGTATAC TAGTTGTCATGCGTCAGCGTGATGAACTATATTCAGCTCTTAAGTTTATAGAGCTTGATA GTGTAGCAAGTCTGCAAGGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTG TTCACCCAGCTTATGCAGTCACAACTAAGGCTCTTGCTGCAACTTATAAAGTTAATGATG AACTTGCTGCACTTGTTAACGTGGAAGCTGGTTCAGAAATAACATATAAACATCTTATTT CTTTGTTAGGGTTTAAGATGAGTGTTAATGTTGAAGGCTGCCACAACATGTTTATAACAC GTGATGAGGCTATCCGCAACGTAAGAGGTTGGGTAGGTTTTGATGTAGAAGCAACACATG CTTGCGGTACTAACATTGGTACTAACCTGCCTTTCCAAGTAGGTTTCTCTACTGGTGCAG ACTTTGTAGTTACGCCTGAGGGACTTGTAGATACTTCAATAGGCAATAATTTTGAGCCTG TGAATTCTAAAGCACCTCCAGGTGAACAATTTAATCACTTGAGAGCGTTATTCAAAAGTG CTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCA ACGTTTCAGATTGTGTAGTGTTTGTCACGTGGTGTCATGGCCTAGAACTAACCACTTTGC GCTATTTTGTTAAAATAGGCAAGGACCAAGTTTGTTCTTGCGGTTCTAGAGCAACAACTT TTAATTCTCATACTCAGGCTTATGCTTGTTGGAAGCATTGCTTGGGTTTTGATTTTGTTT ATAATCCACTCTTAGTGGATATTCAACAGTGGGGTTATTCTGGTAACCTACAATTTAACC ATGATTTGCATTGTAATGTGCATGGACACGCACATGTAGCTTCTGCGGATGCTATTATGA CGCGTTGTCTTGCAATTAATAATGCATTTTGTCAAGATGTCAACTGGGATTTAACTTACC CTCATATAGCAAATGAGGATGAAGTCAATTCTAGCTGTAGATATTTACAACGCATGTATC TTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATAGGCAACCCTAAAG GTATAAAATGTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAATCCAATAG TACCCAATGTCAAGCAGTTTGAGTATGACTATAATCAGCACAAAGATAAGTTTGCTGATG GTCTTTGTATGTTTTGGAATTGTAATGTGGATTGTTATCCCGACAATTCCTTAGTTTGTA GGTACGACACACGAAATTTGAGTGTGTTTAACCTACCTGGTTGTAATGGTGGTAGCTTGT ATGTTAACAAGCATGCATTCCACACACCTAAATTTGATCGCACTAGCTTTCGTAATTTGA AAGCTATGCCATTCTTTTTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAG TTGCGCAAGACCTTGTGTCATTAGCTACGAAAGATTGTATCACAAAATGCAACATAGGCG GTGCTGTTTGTAAAAAGCACGCACAAATGTATGCAGATTTTGTGACTTCTTATAATGCAG CTGTTACTGCTGGTTTTACTTTTTGGGTTACTAATAATTTTAACCCATATAATTTGTGGA AAAGTTTTTCAGCTCTCCAGTCTATCGACAATATTGCTTATAATATGTATAAGGGTGGTC ATTATGATGCTATTGCAGGAGAAATGCCCACTATCGTAACTGGAGATAAAGTTTTTGTTA TAGATCAAGGCGTAGAAAAAGCAGTTTTTTTTAATCAAACAATTCTGCCTAGATCTGTAG CGTTTGAGCTGTATGCGAAGAGAAATATTCGCACACTGCCAAACAACCGTATTTTGAAAG GTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTACACGAACCAAACACCAC TATACCGTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAATGGCCTAATAG TGCTGTATGATGATAGATATGGTGATTACCAGTCTTTTCTAGCTGCTGATAATGCTGTTT TAGTTTCTACACAGTGTTACAAGCGGTATTCGTATGTAGAAATACCGTCAAACCTGCTTG TTCAGAACGGTATTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGCGTGTTAATG GTGCGTTTGTTACGCTACCTAACACATTAAACACACAGGGTCGCAGTTATGAAACTTTTG AACCTCGTAGTGATGTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAAA AGTATGGTAAAGAATTAGGTCTACAGCACATACTGTATGGTGAAGTTGATAAGCCCCAAT TAGGTGGTTTACACACTGTTATAGGTATGTGCAGACTTTTACGTGCGAATAAGTTGAACG CAAAGTCTGTTACTAATTCTGATTCTGATGTCATGCAAAATTATTTTGTATTGGCAGACA ATGGTTCCTACAAGCAAGTGTGTACTGTTGTGGATTTGCTGCTTGATGATTTCTTAGAAC TTCTTAGGAACATACTGAAAGAGTATGGTACTAATAAGTCTAAAGTTGTAACAGTGTCAA TTGATTACCATAGCATAAATTTTATGACTTGGTTTGAAGATGGCATTATTAAAACATGTT ATCCACAGCTTCAATCAGCATGGACGTGTGGTTATAATATGCCTGAACTTTATAAAGTTC AGAATTGTGTTATGGAACCTTGCAACATTCCTAATTATGGTGTTGGAATAGCGTTGCCAA GTGGTATTATGATGAATGTGGCAAAGTATACACAACTCTGTCAATACCTTTCGAAAACAA CAATGTGTGTACCGCATAATATGCGAGTAATGCATTTTGGAGCTGGAAGTGACAAAGGAG TGGCTCCAGGTAGTACTGTTCTTAAACAATGGCTCCCAGAAGGGACACTCCTTGTCGATA ATGATATTGTAGACTATGTGTCTGATGCACATGTTTCTGTGCTTTCAGATTGCAATAAAT ATAAGACAGAGCACAAGTTTGATCTTGTGATATCTGATATGTATACAGACAATGATTCAA AAAGAAAGCATGAAGGCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATATCTCT CAAGTTTTCTTCGTAATAATTTGGCTCTAGGTGGTAGTTTTGCTGTAAAAGTGACAGAGA CAAGTTGGCACGAAGTTTTATATGACATTGCACAGGATTGTGCATGGTGGACAATGTTTT GTACAGCAGTGAATGCCTCTTCTTCAGAAGCATTCTTGGTTGGTGTTAATTATTTGGGTG CAAGTGAAAAGGTTAAGGTTAGTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGA ATTGTAATTATTTACAAACCTCTGCTTATAGTATATTTGACGTTGCTAAGTTTGATTTGA GATTGAAAGCAACACCAGTTGTTAATTTGAAAACTGAACAAAAGAGAGACTTAGTGTTTA ATTTAATTAAGTGTGGTAAGTTACTGGTAAGAGATGTTGGTAACACCTCTTTTACTAGTG TACCAAAGTGCCTTTAGACCACCTAATGGTTGGCATTTACACGGGGGTGCTTATGCGGTA GTTAATATTTCTAGCGAATCTAATAATGCAGGCTCTTCACCTGGGTGTATTGTTGGTACT ATTCATGGTGGTCGTGTTGTTAATGCTTCTTCTATAGCTATGACGGCACCGTCATCAGGT ATGGCTTGGTCTAGCAGTCAGTTTTGTACTGCACACTGTAACTTTTCAGATACTACAGTG TTTGTTACACATTGTTATAAATATGATGGGTGTCCTATAACTGGCATGCTTCAAAAGAAT TTTTTACGTGTTTCTGCTATGAAAAATGGCCAGCTTTTCTATAATTTAACAGTTAGTGTA GCTAAGTACCCTACTTTTAAATCATTTCAGTGTGTTAATAATTTAACATCCGTATATTTA AATGGTGATCTTGTTTACACCTCTAATGAGACCACAGATGTTACATCTGCAGGTGTTTAT TTTAAAGCTGGTGGACCTATAACTTATAAAGTTATGAGAGAAGTTAAAGCCCTGGCTTAT TTTGTTAATGGTACTGCACAAGATGTTATTTTGTGTGATGGATCACCTAGAGGCTTGTTA GCATGCCAGTATAATACTGGCAATTTTTCAGATGGCTTTTATCCTTTTATTAATAGTAGT TTAGTTAAGCAGAAGTTTATTGTCTATCGTGAAAATAGTGTTAATACTACTTTTACGTTA CACAATTTCACTTTTCATAATGAGACTGGCGCCAACCCTAATCCTAGTGGTGTTCAGAAT ATTCAAACTTACCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAATTTTTCCTTT CTGAGTAGTTTTGTTTATAAGGAGTCTAATTTTATGTATGGATCTTATCACCCAAGTTGT AATTTTAGACTAGAAACTATTAATAATGGCTTGTGGTTTAATTCACTTTCAGTTTCAATT GCTTACGGTCCTCTTCAAGGTGGTTGCAAGCAATCTGTCTTTAGTGGTAGAGCAACTTGT TGTTATGCTTATTCATATGGAGGTCCTTCGCTGTGTAAAGGTGTTTATTCAGGTGAGTTA GATCTTAATTTTGAATGTGGACTGTTAGTTTATGTTACTAAGAGCGGTGGCTCTCGTATA CAAACAGCCACTGAACCGCCAGTTATAACTCGACACAATTATAATAATATTACTTTAAAT ACTTGTGTTGATTATAATATATATGGCAGAACTGGCCAAGGTTTTATTACTAATGTAACC GACTCAGCTGTTAGTTATAATTATCTAGCAGACGCAGGTTTGGCTATTTTAGATACATCT GGTTCCATAGACATCTTTGTTGTACAAGGTGAATATGGTCTTACTTATTATTAGGTTAAC CCTTGCGAAGATGTCAACCAGCAGTTTGTAGTTTCTGGTGGTAAATTAGTAGGTATTCTT ACTTCACGTAATGAGACTGGTTCTCAGCTTCTTGAGAACCAGTTTTACATTAAAATCACT AATGGAACACGTCGTTTTAGACGTTCTATTACTGAAAATGTTGGAAATTGCCCTTATGTT AGTTATGGTAAGTTTTGTATAAAACCTGATGGTTCAATTGCCACAATAGTACCAAAACAA TTGGAACAGTTTGTGGCACCTTTACTTAATGTTACTGAAAATGTGCTCATACCTAACAGT TTTAATTTAACTGTTACAGATGAGTACATACAAACGCGTATGGATAAGGTCCAAATTAAT TGTCTGCAGTATGTTTGTGGCAATTCTCTGGATTGTAGAGATTTGTTTCAACAATATGGG CCTGTTTGTGACAACATATTGTCTGTAGTAAATAGTATTGGTCAAAAAGAAGATATGGAA CTTTTGAATTTCTATTCTTCTACTAAACCGGCTGGTTTTAATACACCATTTCTTAGTAAT GTTAGCACTGGTGAGTTTAATATTTCTCTTCTGTTAACAACTCCTAGTAGTCCTAGAAGG CGTTCTTTTATTGAAGACCTTCTATTTACAAGCGTTGAATCTGTTGGATTACCAACAGAT GACGCATACAAAAATTGCACTGCAGGACCTTTAGGTTTTCTTAAGGACCTTGCGTGTGCT CGTGAATATAATGGTTTGCTTGTGTTGCCTCCCATTATAACAGCAGAAATGCAAATTTTG TATACTAGTTCTCTAGTAGCTTCTATGGCTTTTGGTGGTATTACTGCAGCTGGTGCTATA CCTTTTGCCACACAACTGCAGGCTAGAATTAATCACTTGGGTATTACCCAGTCACTTTTG TTGAAGAATCAAGAAAAAATTGCTGCTTCCTTTAATAAGGCCATTGGTCGTATGCAGGAA GGTTTTAGAAGTACATCTCTAGCATTACAACAAATTCAAGATGTTGTTAATAAGCAGAGT GCTATTCTTACTGAGACTATGGCATCACTTAATAAAAATTTTGGTGCTATTTCTTCTATG ATTCAAGAAATCTACCAGCAACTTGACGCCATACAAGCAAATGCTCAAGTGGATCGTCTT ATAACTGGTAGATTGTCATCACTTTCTGTTTTAGCATCTGCTAAGCAGGCGGAGCATATT AGAGTGTCACAACAGCGTGAGTTAGCTACTCAGAAAATTAATGAGTGTGTTAAGTCACAG TCTATTAGGTACTCCTTTTGTGGTAATGGACGACATGTTCTAACCATACCGCAAAATGCA CCTAATGGTATAGTGTTTATACACTTTTCTTATACTCCAGATAGTTTTGTTAATGTTACT GCAATAGTGGGTTTTTGTGTAAAGCCAGCTAATGCTAGTCAGTATGCAATAGTACCCGCT AATGGTAGGGGTATTTTTATACAAGTTAATGGTAGTTACTACATCACAGCACGAGATATG TATATGCCAAGAGCTATTACTGCAGGAGATATAGTTACGCTTACTTCTTGTCAAGCAAAT TATGTAAGTGTAAATAAGACCGTCATTACTACATTCGTAGACAATGATGATTTTGATTTT AATGACGAATTGTCAAAATGGTGGAATGACACTAAGCATGAGCTACCAGACTTTGACAAA TTCAATTACACAGTACCTATACTTGACATTGATAGTGAAATTGATCGTATTCAAGGCGTT ATACAGGGTCTTAATGACTCTTTAATAGACCTTGAAAAACTTTCAATACTCAAAACTTAT ATTAAGTGGCCTTGGTATGTGTGGTTAGCCATAGCTTTTGCCACTATTATCTTCATCTTA ATACTAGGATGGGTTTTCTTCATGACTGGATGTTGTGGTTGTTGTTGTGGATGCTTTGGC ATTATGCCTCTAATGAGTAAGTGTGGTAAGAAATCTTCTTATTACACGACTTTTGATAAC GATGTGGTAACTTAACAATACAGACCTAAAAAGTCTGTTTAATGATTCAAAGTCCCACGT CCTTCCTAATAGTATTAATTTTTCTTTGGTGTAAACTTGTACTAAGTTGTTTTAGAGAGT TTATTATAGCGCTCCAACAACTAATACAAGTTTTACTCCAAATTATCAATAGTAACTTAC AGCCTAGACTGACCCTTTGTCACAGTCTAGACTAATGTTAAACTTAGAAGCAATTATTGA AACTGGTGAGCAAGTGATTCAAAAAATCAGTTTCAATTTACAGCATATTTCAAGTGTATT AAACACAGAAGTATTTGACCCCTTTGACTATTGTTATTACAGAGGAGGTAATTTTTGGGA AATAGAGTCAGCTGAAGATTGTTCAGGTGATGATGAATTTATTGAATAAGTCGCTAGAGG AAAATGGAAGTTTTCTAACAGCGCTTTATATATTTGTAGGATTTTTAGCACTTTATCTTC TAGGTAGAGCACTTCAAGCATTTGTACAGGCTGCTGATGCTTGTTGTTTATTTTGGTATA CATGGGTAGTAATTCCAGGAGCTAAGGGTACAGCCTTTGTATATAAGTATACATATGGTA GAAAACTTAACAATCGGGAATTAGAAGCAGTTATTGTCAACGAGTTTCCTAAGAACGGTT GGAATAATAAAAATCCAGCAAATTTTCAAGATGTCCAACGAGACAAATTGTACTCTTGAC TTTGAACAGTCAGTTGAGCTTTTTAAAGAGTATAATTTATTTATAACTGCATTCTTGTTG TTCTTAACCATAATACTTCAGTATGGCTATGCAACAAGAAGTAAGTTTATTTATATACTG AAAATGATAGTGTTATGGTGCTTTTGGCCCCTTAACATTGCAGTAGGTGTAATTTCATGT ATATACCCACCAAACACAGGAGGTCTTGTCGCAGCGATAATACTTACAGTGTTTGCGTGT CTGTCTTTTGTAGGTTATTGGATCCAGAGTATTAGACTCTTTAAGCGGTGTAGGTCATGG TGGTCATTTAACCCAGAATCTAATGCCGTAGGTTCAATACTCCTAACTAATGGTCAACAA TGTAATTTTGCTATAGAGAGTGTGCCAATGGTGCTTTCTCCAATTATAAAGAATGGTGTT CTTTATTGTGAGGGTCAGTGGCTTGCTAAGTGTGAACCAGACCACTTGCCTAAAGATATA TTTGTTTGTACACCGGATAGACGTAATATCTACCGTATGGTGCAGAAATATACTGGTGAC CAAAGCGGAAATAAGAAACGGTTTGCTACGTTTGTCTATGCAAAGCAGTCAGTAGATACT GGCGAGCTAGAAAGTGTAGCAACAGGAGGGAGTAGTCTTTACACCTAAATGTGTGTGTGT AGAGAGTATTTAAAATTATTCTTTAATAGTGCCTCTATTTTAAGAGCGCATAATAGTATT ATTTTTGAGGATATTAATATAAATCCTCTCTGTTTTATACTCTCTTTTCAAGAGCTATTA TTTAAAAAACAGTTTTTCCACTCTTTTGTGCCAAAAACTATTGTTGTTAATGGTGTAACC TTTCAAGTAGATAATGGAAAAGTCTACTACGAAGGAAAACCAATTTTTCAGAAAGGTTGT TGTAGGTTGTGGTTGAGTTATAAAAAAGATTAAACTACCTACTACACTTATTTTTATAAG AGGCGTTTTATCTTACAAGCGCTTAATAAATACGGACGATGAAATGGCTGACTAGTTTTG TAAGGGCAGTTATTTCATGTTATAAACCCCTATTATTAACTCAATTAAGAGTATTAGATA GGTTAATCTTAGATCATGGACCAAAACACATCTTAACGTGTGTTAGGTGCGTGATTTTGT TTCAATTAGATTTAGTTTATAGGTTGGCGTATACGCCTACTCAATCGCTGGTATGAATAA TAGTAAAGATAATCCTTTTTGCGGAGCAATAGCAAGAAAAGCGCGAATTTATCTGAGAGA AGGATTAGATTGTGTTTACTTTCTTAACAAAGCAGGACAAGCAGAGTCTTGTCCCGCGTG TACCTCTCTAGTATTCCAGGGGAAAACTTGTGAGGAACACAAATATAATAATAATCTTTT GTCATGGCAAGCGGTAAGGCAACTGGAAAGACAGATGCCCCAGCTCCAGTCATCAAACTA GGAGGACCAAAGCCACCTAAAGTTGGTTCTTCTGGAAATGTATCTTGGTTTCAAGCAATA AAAGCCAAGAAGTTAAATTCACCTCCGCCTAAGTTTGAAGGTAGCGGTGTTCCTGATAAT GAAAATCTAAAACCAAGTCAGCAGCATGGATATTGGAGACGCCAAGCTAGGTTTAAGCCA GGTAAAGGTGGAAGAAAACCAGTCCCAGATGCTTGGTATTTTTAGTATACTGGAACAGGA CCAGCCGCTAACCTGAATTGGGGTGATAGCCAAGATGGTATAGTGTGGGTTGCTGGTAAG GGTGCTGATACTAAATTTAGATCTAATCAGGGTACTCGTGACTCTGACAAGTTTGACCAA TATCCGCTACGGTTTTCAGACGGAGGACCTGATGGTAATTTCCGTTGGGATTTCATTCCT CTGAATCGTGGCAGGAGTGGGAGATCAACAGCAGCTTCATCAGCAGCATCTAGTAGAGCA CCATCACGTGAAGTTTCGCGTGGTCGCAGGAGTGGTTCTGAAGATGATCTTATTGCTCGT GCAGCAAGGATAATTCAGGATCAGCAGAAGAAGGGTTCTCGCATTACAAAGGCTAAGGCT GATGAAATGGCTCACCGCCGGTATTGCAAGCGCAGTATTCCACCTAATTATAAGGTTGAT CAAGTGTTTGGTCCCCGTACTAAAGGTAAGGAGGGAAATTTTGGTGATGACAAGATGAAT GAGGAAGGTATTAAGGATGGGCGCGTTACAGCAATGCTCAACCTAGTTCCTAGCAGCCAT GCTTGTCTTTTCGGAAGTAGAGTGACGCCCAGACTTCAACCAGATGGGCTGCACTTGAAA TTTGAATTTACTACTGTGGTCCCACGTGATGATCCGCAGTTTGATAATTATGTAAAAATT TGTGATCAGTGTGTTGATGGTGTAGGAACACGTCCAAAAGATGATGAACCAAGACCAAAG TCACGCTCAAGTTCAAGACCTGCAACAAGAGGAAATTCTCCAGCGCCAAGACAGCAGCGC CCTAAGAAGGAGAAAAAGCCAAAGAAGCAGGATGATGAAGTGGATAAAGCATTGACCTCA GATGAGGAGAGGAACAATGCACAGCTGGAATTTGATGATGAACCCAAGGTAATTAACTGG GGGGATTCAGCGCTAGGAGAGAATGAACTTTGAGTAAAATTGAATAGTAAGAGTTAAGGA AGATAGGCATGTAGCTTGATTACCTACATGTCTATCGCCAGGGAAATGTCTAATTTGTCT ACTTAGTAGCCTGGAAACGAACGGTAGACCCTTAGATTTTAATTTAGTTTAATTTTTAGT TTAGTTTAAGTTAGTTTAGAGTAGGTATAAAGATGCCAGTGGCGGGGCCACGCGGAGTAC GACCGAGGGTACAGCACTAGGACGCCCATTAGGGGAAGAGCTAAATTTTAGTTTAAGTTA AGTTTAATTGGCTATGTATAGTTAAAATTTATAGGCTAGTATAGAGTTAGAGCAAAAAAA AAAAAAAAAAAAAAAAAAAA

Replicase

In addition to the structural and accessory genes, two-thirds of a coronavirus genome comprises the replicase gene (at the 5′ end of the genome), which is expressed as two polyproteins, pp1a and pp1ab, in which pp1ab is an extension product of pp1a as a result of a −1 ribosomal shift mechanism. The two polyproteins are cleaved by two types of virus-encoded proteinases usually resulting in 16 non-structural proteins (Nsp1-16); IBV lacks Nsp1 thereby encoding Nsp2-16.

Thus Gene 1 in IBV encodes 15 (16 in other coronaviruses) non-structural proteins (nsp2-16), which are associated with RNA replication and transcription.

The term ‘replicase protein’ is used herein to refer to the pp1a and pp1ab polyproteins or individual nsp subunits.

The term ‘replicase gene’ is used herein to refer to a nucleic acid sequence which encodes for replicase proteins.

A summary of the functions of coronavirus nsp proteins is provided in Table 1.TABLE 1 Nsp Protein Key features 1 Conserved within but not between coronavirus genetic groups; potential regulatory functions in the host cell. 2 Dispensable for MHV and SARS-CoV replication in tissue culture 3 Acidic domain; macro domain with ADRP and poly (ADP-ribose)-binding activities; one or two ZBD- containing papain-like proteases; Y domain 4 Transmembrane domain 5 3C-like main protease, homodimer 6 Transmembrane domain 7 Interacts with nsp8 to form a hexadecamer complex 8 Noncannonical RNA polymerase; interacts with nsp7 to form a hexadecameric complex 9 ssRNA-binding protein, dimer 10 RNA-binding protein, homododecamer, zinc-binding domain, known to interact with nsp14 and nsp16 11 Unknown 12 RNA-dependent RNA polymerase 13 Zinc-binding domain, NTPase, dNTPase, 5′-to-3′ RNA and DNA helicase, RNA 5′-triphosphate 14 3′-to 5′ exoribonuclease, zinc-binding domain and N7- methyltransferase 15 Uridylate-specific endoribonuclease, homohexamer 16 Putative ribose-2′-O-methyltransferase

The variant replicase gene encoded by the coronavirus of the present invention comprises a mutation in one or more of the sections of sequence encoding nsp-10, nsp-14, nsp-15 or nsp-16.

Nsp10 has RNA-binding activity and appears to be involved in homo and/or heterotypic interactions within other nsps from the pp1a/pp1ab region. It adopts an α/β fold comprised of five α-helices, one 310-helix and three β-strands. Two zinc-binding sites have been identified that are formed by conserved cysteine residues and one histidine residue (Cys-74/Cys-77/His-83/Cys-90; Cys-117/Cys-120/Cys-128/Cys-130). The protein has been confirmed to bind single-stranded and double-stranded RNA and DNA without obvious specificity. Nsp-10 can be cross-linked with nsp-9, suggesting the existing of a complex network of protein-protein interactions involving nsp-7, -8, -9 and -10. In addition, nsp-10 is known to interact with nsp-14 and nsp-16.

Nsp-14 comprises a 3′-to-5′ exoribonuclease (ExoN) active domain in the amino-terminal region. SARS-CoV ExoN has been demonstrated to have metal ion-dependent 3′-to-5′ exoribonuclease activity that acts on both single-stranded and double-stranded RNA, but not on DNA. Nsp-14 has been shown to have proof-reading activity. This nsp has also been shown to have N7-methyltransferase (MT) activity in the carboxyl-terminal region.

Nsp-15 associated NendoU (nidoviral endoribonuclease, specific for U) RNase activity has been reported for a number of coronaviruses, including SARS-CoV, MHV and IBV. The activities were consistently reported to be significantly enhanced by Mn2+ ions and there was little activity in the presence of Mg2+ and Ca2+. NendoU cleaves at the 3′ side of uridylate residues in both single-stranded and double-stranded RNA. The biologically relevant substrate(s) of coronavirus NendoUs remains to be identified.

Nsp-16 has been predicted to mediate ribose-2′-O-methyltransferase (2′-O-MTase) activity and reverse-genetics experiments have shown that the 2′-O-MTase domain is essential for viral RNA synthesis in HCoV-229E and SARS-CoV. The enzyme may be involved in the production of the cap 1 structures of coronavirus RNAs and it may also cooperate with NendoU and ExoN in other RNA processing pathways. 2′-O-MTase might also methylate specific RNAs to protect them from NendoU-mediated cleavage.

The genomic and protein sequences for nsp-10, -14, -15 and -16 are provided as SEQ ID NO: 2-5 and 6-9, respectively.(nsp-10 nucleotide sequence- nucleotides 11884-12318 of SEQ ID NO: 1) SEQ ID NO: 2 TCTAAAGGTCATGAGACAGAGGAAGTGGATGCTGTAGGCATTCTCTCACTTTGTTCTTTTGCAGTA GATCCTGCGGATACATATTGTAAATATGTGGCAGCAGGTAATCAACCTTTAGGTAACTGTGTTAAA ATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAGCCAAGTCCAACTCCGGATCAG GATTCTTATGGAGGAGCTTCTGTGTGTCTTTATTGTAGAGCACATATAGCACACCTTGGCGGAGCA GGAAATTTAGATGGACGCTGTCAATTTAAAGGTTCTTTTGTGCAAATACCTACTACGGAGAAAGAT CCTGTTGGATTCTGTCTACGTAACAAGGTTTGCACTGTTTGTCAGTGTTGGATTGGTTATGGATGT CAGTGTGATTCACTTAGACAACCTAAACCTTCTGTTCAG (nsp-14 nucleotide sequence- nucleotides 16938-18500 of SEQ ID NO: 1) SEQ ID NO: 3 GGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTGTTCACCCAGCTTATGCAGTCACA ACTAAGGCTCTTGCTGCAACTTATAAAGTTAATGATGAACTTGCTGCACTTGTTAACGTGGAAGCT GGTTCAGAAATAACATATAAACATCTTATTTCTTTGTTAGGGTTTAAGATGAGTGTTAATGTTGAA GGCTGCCACAACATGTTTATAACACGTGATGAGGCTATCCGCAACGTAAGAGGTTGGGTAGGTTTT GATGTAGAAGCAACACATGCTTGCGGTACTAACATTGGTACTAACCTGCCTTTCCAAGTAGGTTTC TCTACTGGTGCAGACTTTGTAGTTACGCCTGAGGGACTTGTAGATACTTCAATAGGCAATAATTTT GAGCCTGTGAATTCTAAAGCACCTCCAGGTGAACAATTTAATCACTTGAGAGCGTTATTCAAAAGT GCTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCAACGTT TCAGATTGTGTAGTGTTTGTCACGTGGTGTCATGGCCTAGAACTAACCACTTTGCGCTATTTTGTT AAAATAGGCAAGGACCAAGTTTGTTCTTGCGGTTCTAGAGCAACAACTTTTAATTCTCATACTCAG GCTTATGCTTGTTGGAAGCATTGCTTGGGTTTTGATTTTGTTTATAATCCACTCTTAGTGGATATT CAACAGTGGGGTTATTCTGGTAACCTACAATTTAACCATGATTTGCATTGTAATGTGCATGGACAC GCACATGTAGCTTCTGCGGATGCTATTATGACGCGTTGTCTTGCAATTAATAATGCATTTTGTCAA GATGTCAACTGGGATTTAACTTACCCTCATATAGCAAATGAGGATGAAGTCAATTCTAGCTGTAGA TATTTACAACGCATGTATCTTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATA GGCAACCCTAAAGGTATTAAATGTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAAT CCAATAGTACCCAATGTCAAGCAGTTTGAGTATGACTATAATCAGCACAAAGATAAGTTTGCTGAT GGTCTTTGTATGTTTTGGAATTGTAATGTGGATTGTTATCCCGACAATTCCTTACTTTGTAGGTAC GACACACGAAATTTGAGTGTGTTTAACCTACCTGGTTGTAATGGTGGTAGCTTGTATGTTAACAAG CATGCATTCCACACACCTAAATTTGATCGCACTAGCTTTCGTAATTTGAAAGCTATGCCATTCTTT TTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAGTTGCGCAAGACCTTGTGTCATTA GCTACGAAAGATTGTATCACAAAATGCAACATAGGCGGTGCTGTTTGTAAAAAGCACGCACAAATG TATGCAGATTTTGTGACTTCTTATAATGCAGCTGTTACTGCTGGTTTTACTTTTTGGGTTACTAAT AATTTTAACCCATATAATTTGTGGAAAAGTTTTTCAGCTCTCCAG (nsp-15 nucleotide sequence- nucleotides 18501-19514 of SEQ ID NO: 1) SEQ ID NO: 4 TCTATCGACAATATTGCTTATAATATGTATAAGGGTGGTCATTATGATGCTATTGCAGGAGAAATG CCCACTATCGTAACTGGAGATAAAGTTTTTGTTATAGATCAAGGCGTAGAAAAAGCAGTTTTTTTT AATCAAACAATTCTGCCTACATCTGTAGCGTTTGAGCTGTATGCGAAGAGAAATATTCGCACACTG CCAAACAACCGTATTTTGAAAGGTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTAC ACGAACCAAACACCACTATACCGTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAAT GGCCTAATAGTGCTGTATGATGATAGATATGGTGATTACCAGTCTTTTCTAGCTGCTGATAATGCT GTTTTAGTTTCTACACAGTGTTACAAGCGGTATTCGTATGTAGAAATACCGTCAAACCTGCTTGTT CAGAACGGTATTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGCGTGTTAATGGTGCGTTT GTTACGCTACCTAACACAATAAACACACAGGGTCGAAGTTATGAAACTTTTGAACCTCGTAGTGAT GTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAAAAGTATGGTAAAGAATTAGGT CTACAGCACATACTGTATGGTGAAGTTGATAAGCCCCAATTAGGTGGTTTCCACACTGTTATAGGT ATGTGCAGACTTTTACGTGCGAATAAGTTGAACGCAAAGTCTGTTACTAATTCTGATTCTGATGTC ATGCAAAATTATTTTGTATTGGCAGACAATGGTTCCTACAAGCAAGTGTGTACTGTTGTGGATTTG CTGCTTGATGATTTCTTAGAACTTCTTAGGAACATACTGAAAGAGTATGGTACTAATAAGTCTAAA GTTGTAACAGTGTCAATTGATTACCATAGCATAAATTTTATGACTTGGTTTGAAGATGGCATTATT AAAACATGTTATCCACAGCTTCAA (nsp-16 nucleotide sequence- nucleotides 19515-20423 of SEQ ID NO: 1) SEQ ID NO: 5 TCAGCATGGACGTGTGGTTATAATATGCCTGAACTTTATAAAGTTCAGAATTGTGTTATGGAACCT TGCAACATTCCTAATTATGGTGTTGGAATAGCGTTGCCAAGTGGTATTATGATGAATGTGGCAAAG TATACACAACTCTGTCAATACCTTTCGAAAACAACAATGTGTGTACCGCATAATATGCGAGTAATG CATTTTGGAGCTGGAAGTGACAAAGGAGTGGTGCCAGGTAGTACTGTTCTTAAACAATGGCTCCCA GAAGGGACACTCCTTGTCGATAATGATATTGTAGACTATGTGTCTGATGCACATGTTTCTGTGCTT TCAGATTGCAATAAATATAAGACAGAGCACAAGTTTGATCTTGTGATATCTGATATGTATACAGAC AATGATTCAAAAAGAAAGCATGAAGGCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATAT CTCTCAAGTTTTCTTCGTAATAATTTGGCTCTAGGTGGTAGTTTTGCTGTAAAAGTGACAGAGACA AGTTGGCACGAAGTTTTATATGACATTGCACAGGATTGTGCATGGTGGACAATGTTTTGTACAGCA GTGAATGCCTCTTCTTCAGAAGCATTCTTGATTGGTGTTAATTATTTGGGTGCAAGTGAAAAGGTT AAGGTTAGTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGAATTGTAATTATTTACAAACC TCTGCTTATAGTATATTTGACGTTGCTAAGTTTGATTTGAGATTGAAAGCAACGCCAGTTGTTAAT TTGAAAACTGAACAAAAGACAGACTTAGTCTTTAATTTAATTAAGTGTGGTAAGTTACTGGTAAGA GATGTTGGTAACACCTCTTTTACTAGTGACTCTTTTGTGTGTACTATGTAG (nsp-10 amino acid sequence) SEQ ID NO: 6 SKGHETEEVDAVGILSLCSFAVDPADTYCKYVAAGNQPLGNCVKMLTVKNGSGFAITSKPSPTPDQ DSYGGASVCLYCRAHIAHPGGAGNLDGRCQFKGSFVQIPTTEKDPVGFCLRNKVCTVCQCWIGYGC QCDSLRQPKPSVQ (nsp-14 amino acid sequence) SEQ ID NO: 7 GTGLFKICNKEFSGVHPAYAVTTKALAATYKVNDELAALVNVEAGSEITYKHLISLLGFKMSVNVE GCHNMFITRDEAIRNVRGWVGFDVEATHACGTNIGTNLPFQVGFSTGADFVVTPEGLVDTSIGNNF EPVNSKAPPGEQFNHLRALFKSAKPWHVVRPRIVQMLADNLCNVSDCVVFVTWCHGLELTTLRYFV KIGKDQVCSCGSRATTFNSHTQAYACWKHCLGFDFVYNPLLVDIQQWGYSGNLQFNHDLHCNVHGH AHVASADAIMTRCLAINNAFCQDVNWDLTYPHIANEDEVNSSCRYLQRMYLNACVDALKVNVVYDI GNPKGIKCVRRGDLNFRFYDKNPIVPNVKQFEYDYNQHKDKFADGLCMFWNCNVDCYPDNSLVCRY DTRNLSVFNLPGCNGGSLYVNKHAFHTPKFDRTSFRNLKAMPFFFYDSSPCETIQLDGVAQDLVSL ATKDCITKCNICGAVCKKKAQMYADFVTSYNAAVTAGFTFWVTNNFNPYNLWKSFSALQ (nsp-15 amino acid sequence) SEQ ID NO: 8 SIDNIAYNMYKGGHYDAIAGEMPTIVTGDKVFVIDQGVEKAVFFNQTILPTSVAFELYAKRNIRTL PNNRILKGLGVDVTNGFVIWDYTNQTPLYRNTVKVCAYTDIEPNGLIVLYDDRYGDYQSFLAADNA VLVSTQCYKRYSYVEIPSNLLVQNGIPLKDGANLYVYKRVNGAFVTLPNTLNTQGRSYETFEPRSD VERDFLDMSEESFVEKYGKELGLQHILYGEVDKPQLGGLHTVIGMCRLLRANKLNAKSVTNSDSDV MQNYFVLADNGSYKQVCTVVDLLLDDFLELLRNILKEYGTNKSKVVTVSIDYHSINFMTWFEDGII KTCYPQLQ (nsp-16 amino acid sequence) SEQ ID NO: 9 SAWTCGYNMPELYKVQNCVMEPCNIPNYGVGIALPSGIMMNVAKYTQLCQYLSKTTMCVPHNMRVM HFGAGSDKGVAPGSTVLKQWLPEGTLLVDNDIVDYVSDAHVSVLSDCNKYKTEHKFDLVISDMYTD NDSKRKHEGVIANNGNDDVFIYLSSFLRNNLALGGSFAVKVTETSWHEVLYDIAQDCAWWTMFCTA VNASSSEAFLVGVNYLGASEKVIWSGKTLHANYIFWRNCNYLQTSAYSIFDVAKFDLRLKATPVVN LKTEQKTDLVFNLIKCGKLLVRDVGNTSFTSDSFVCTM

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.EXAMPLESExample 1—Generation of an IBV Reverse Genetics System Based on M41-CK

A M41-CK full-length cDNA was produced by replacement of the Beaudette cDNA in the Vaccinia virus reverse genetics system previously described in PCT/GB2010/001293 (herein incorporated by reference) with synthetic cDNA derived from the M41 consensus sequence.

The IBV cDNA within recombinant Vaccinia virus (rVV) rVV-BeauR-Rep-M41 structure described in Armesto, Cavanagh and Britton (2009). PLoS ONE 4(10): e7384. doi:10.1371/journal.pone.0007384, which consisted of the replicase derived from IBV Beaudette strain and the structural and accessory genes and 3′ UTR from IBV M41-CK, was further modified by replacement of the Beaudette 5′ UTR-Nsp2-Nsp3 sequence with the corresponding sequence from IBV M41-CK. The resulting IBV cDNA consisted of 5′ UTR-Nsp2-Nsp3 from M41, Nsp4-Nsp16 from Beaudette and the structural and accessory genes and 3′ UTR from M41. This cDNA was further modified by the deletion of the Beaudette Nsp4-Nsp16 sequence. The resulting cDNA, lacking Nsp4-16, was modified in four further steps in which the deleted Nsps were sequentially replaced with the corresponding sequences from M41-CK, the replacement cDNAs represented M41-CK Nsp4-8, Nsp9-12, Nsp12-14 and finally Nsp15-16. Each replacement cDNA contained approx. 500 nucleotides at the 5′ end corresponding to the 3′ most M41 sequence previously inserted and approx. 500 nucleotides at the 3′ end corresponding to the M41 S gene sequence. This allowed insertion of the M41 cDNA sequence by homologous recombination and sequential addition of contiguous M41 replicase gene sequence. The synthetic cDNAs containing the M41-derived Nsp sequences were added by homologous recombination utilising the inventor’s previous described transient dominant selection (IDS) system (see PCT/GB2010/001293). The M41-derived cDNAs containing sequence corresponding to the M41 Nsps-10, -14, -15 and -16 contained the modified amino acids at positions 85, 393, 183 and 209, respectively, as indicated in FIG. 10.

A full-length cDNA representing the genome of M41-CK was generated in Vaccinia virus representing the synthetic sequences. Two rIBVs, M41-R-6 and M41-R-12, were rescued and shown to grow in a similar manner as M41-CK (FIG. 1).Example 2—Determining the Pathogenicity of Rescued M41 Viruses

The viruses rescued in Example 1 were used to infect 8-day-old specific pathogen free (SPF) chicks by ocular and nasal inoculation to test them for pathogenicity, as observed by clinical signs on a daily basis 3-7 days post-infection and for ciliary activity days 4 and 6 post-infection. Loss of ciliary activity is a well-established method for determining the pathogenicity of IBV. The two M41-R viruses were found to be apathogenic when compared to M41-CK though they did show some clinical signs in comparison to uninfected control chicks (FIG. 2) and some but inconsistent loss in ciliary activity (FIG. 3).

Thus, the M41-R molecular clones of M41-CK were not pathogenic when compared to the parental virus M41-CK.

The inventors identified several nucleotide differences in the M41-R compared to the M41-CK sequences. The majority of these were synonymous mutations, as the nucleotide change did not affect the amino acid sequence of the protein associated with the sequence. However, four non-synonymous mutations were identified in the IBV replicase gene specific to Nsp-10, Nsp-14, Nsp-15 and Nsp-16 components of the replicase gene, these mutations resulted in amino acid changes (Table 3).TABLE 3 Non-Synonymous mutations identified in the Nsps of M41-R full-length genome Region of Nucleotide Nucleotide Replicase position Mutation Amino Acid Change Nsp10 12137 C→T Pro→Leu Nsp14 18114 G→C Val→Leu Nsp15 19047 T→A Leu→Ile Nsp16 20139 G→A Val→Ile

Example 3—Repair of M41-R rIBVs

In order to determine whether the identified mutations were responsible for the loss of pathogenicity associated with M41-R, the Nsp10 mutation was repaired and the mutations in Nsp-14, -15 & -16 were repaired and shown to grow in a similar manner as M41-CK (FIG. 9). The inventors thus generated the rIBVs, M41R-nsp10rep and M41R-nsp14, 15, 16rep, using synthetic cDNAs containing the correct nucleotides utilising the inventor’s previous described (TDS) system (see PCT/GB2010/001293).

The rIBVs were assessed for pathogenicity in chicks as described previously. Both rIBVs showed increased pathogenicity when compared to M41-R but not to the level observed with M41-CK (FIGS. 4 and 5). M41R-nsp14, 15, 16rep gave more clinical signs and more reduction in ciliary activity than M41R-nsp10rep, overall these results indicated that the changes associated with the four Nsps appear to affect pathogenicity.

To determine the roles of the Nsps in pathogenicity the full-length cDNA corresponding to M41R-nsp10rep was used to repair the mutations in Nsps14, 15 & 16 using a synthetic cDNA containing the correct nucleotides utilising the TDS system.

The following rIBVs were produced:

M41R-nsp10, 15rep—M41-R with the mutations in Nsp-10 and Nsp-15 repaired

M41R-nsp10, 14, 15rep—M41-R with mutations in Nsp-10, -14 and -15 repaired

M41R-nsp10, 14, 16rep—M41-R with mutations in Nsp-10, -14 and -16 repaired

M41R-nsp10, 15, 16rep—M41-R with mutations in Nsp-10, -15 and -16 repaired

M41-K—All four mutations, Nsp-10, -14, -15 & -16 repaired in M41-R

The rIBVs were shown to grow in a similar manner as M41-CK (FIG. 9) and assessed for pathogenicity as described previously. M41-K (in which all four mutations had been repaired) resulted in clinical signs and 100% loss of ciliary activity (complete ciliostasis) by 4 days post-infection (FIGS. 6, 7 & 8). The other rIBVs demonstrated varying levels of pathogenicity, apart from M41R-nsp10, 15, 16rep, which was essentially apathogenic. These results confirmed that repair of all four Nsps restored pathogenicity to M41-R; again supporting the previous evidence that the mutations described in the four Nsps are implicated in attenuating M41-CK.

The inventors also generated rIBV M41R-nsp 10, 14 rep (nsp 10 and 14 are repaired, nsp 15 and 16 contain mutations) and rIBV M41R-nsp 10, 16 rep (nsp 10 and 16 are repaired, nsp 14 and 15 contain mutations) and assessed the pathogenicity of these viruses.

rIBV M41R-nsp 10, 14 rep less pathogenic than M41-K but caused around 50% ciliostasis on days 4-6 post-infection. rIBV M41R-nsp 10, 16 rep was almost apathogenic and caused no ciliostasis (see FIG. 11ac).

Thus the genome associated with M41-R is a potential backbone genome for a rationally attenuated IBV.Example 4—Vaccination/Challenge Study with M41-R

Candidate vaccine viruses were tested in studies in which fertilized chicken eggs were vaccinated in ovo at 18 days embryonation and in which the hatchability of the inoculated eggs was determined. The clinical health of the chickens was investigated and the chickens were challenged at 21 days of age with a virulent IB M41 challenge virus at 103.65 EID50 per dose.

Clinical signs were investigated after challenge protection by the vaccine and a ciliostasis test was performed at 5 days after challenge to investigate the effect of the challenge viruses on movement of the cilia and protection by the vaccine against ciliostasis (inhibition of cilia movement).

In Ovo Vaccination in Commercial Broiler Eggs

The design of the experiment is given in Table 4 and the clinical results are given in Table 5. Hatchability of the eggs inoculated with IB M41-R was good and chickens were healthy. IB M41-R protected against clinical signs after challenge in the broilers (placebo: 19/19 affected, 1B M41-R: 3/18 affected and 1 dead). The results of the ciliostasis test are given in Table 6. IB M41-R generated protection against ciliostasis.TABLE 4 Design of a hatchability, safety, efficacy study in commercial eggs EID501 Route Day(s) Day(s) End Nr. of Treatment per of of of of eggs per Treatment Description dose Admin Admin Challenge2 Study treatment T01 None NA NA NA NA NA 30 T02 IB M41-R 104 In ovo 18 days At 21 days At 26 30 NTX Saline NA In ovo embryo- of age, 20 days 30 nation chickens of age per group 1Dose volume 0.1 ml, NA, not applicable. 2103.65 EID50 per dose.

TABLE 5 Hatch percentages and clinical data before and after challenge in commercial chickens, for design see Table 1. Before After challenge challenge Hatch/ Vital/ Deaths/ Symptoms/ Deaths/ Symptoms/ Treatment total total total total total total None 28/30 Euthanized directly after hatch for blood collection IB M41-R 28/30 28/28 1/20 0/19 1/19  3/181, 7 Saline 29/30 29/29 1/20 0/19 0/19 19/191, 2, 3, 4, 5, 6, 71Disturbed respiratory system 2Whizzing 3Change of voice 4Breathing difficult 5Swollen intra-orbital sinuses 6Uneven growth 7Weak

TABLE 6 Results of the ciliostasis test after challenge, for design see Table 1. Treatment Protected/total Percentage protection Saline 0/19  0% IB M41R 5/18 28%

In Ovo Vaccination in Specific Pathogen-Free (SPF) Eggs

The design of the study in SPF eggs is given in Table 7 and is similar with the design of the studies with commercial broilers, but the vaccination dose for 1B M41-R was higher, (10EID50 per dose).

The results (Table 8) show that the hatch percentage for IB M41-R hatch was low, and 19 of 40 hatched and the chicks were weak. Eight chicks died. The remaining 11 chickens were challenged and 11 of the chicks hatched from the eggs which had been inoculated with saline were challenged.

In the ciliostasis test after challenge it appeared that all chickens vaccinated in ovo with IB M41-R were protected, whereas none of the controls was protected, see Table 9.TABLE 7 Design of a hatchability, safety, efficacy study in SPF eggs EID501 Route Day Day End Nr. of Treatment per of of of of eggs per Treatment Description dose Admin Admin Challenge2 Study treatment T01 IB M41-R 105 In ovo 18 days At 21 days At 26 40 embryo- of age days T04 Saline NA In ovo nation of age 40 NTX NA NA NA NA 10 1Dose volume 0.1 ml, NA, not applicable. 2Challenge dose 103.3 EID50 in 0.2 ml.

TABLE 8 Hatch percentages and clinical data before and after challenge in SPF chickens, for design see Table 7. Before After challenge challenge Hatch/ Vital/ Deaths/ Symptoms/ Deaths/ Symptoms/ Treatment total total total total total total IB M41-R 19/40 11/40 8/40 weak 0 0 Saline 30/40 30/40 0 — 0 0 NA  9/10  9/10 0 — — —

TABLE 9 Results of the ciliostasis test after challenge, for design see Table 7. Treatment Protected/total Percentage protection Saline  0/11  0% IB M41R 11/11 100%

In conclusion, IB M41-R was safe in commercial eggs, generated protection against clinical signs and to an extent against ciliostasis.

In SPF eggs vaccinated with IB M41 R a relatively low number of chickens hatched. This may be due to the 10EID50 per egg of 1B M41-R used. This was 10-fold higher than the dose used in earlier studies in which there was a higher level of hatchability. The lower hatch percentages may also be caused by a particularly high susceptibility of the batch of SPF eggs for viruses, as in other studies the level of embryo mortality was also higher that had previously been observed.

After challenge all surviving chickens after hatch were completely protected against ciliostasis. It is concluded that IB M41-R has great potential as vaccine to be administered in ovo.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, virology or related fields are intended to be within the scope of the following claims.

Claims

1. A live, attenuated coronavirus comprising a variant replicase gene encoding polyproteins comprising a mutation in one or both of non-structural protein(s) nsp-10 and nsp-14, wherein the variant replicase gene encodes a protein comprising an amino acid mutation of Pro to Leu at the position corresponding to position 85 of SEQ ID NO: 6, and/or wherein the variant replicase gene encodes a protein comprising an amino acid mutation of Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7.

2. The coronavirus according to claim 1 wherein the variant replicase gene encodes a protein comprising one or more amino acid mutations selected from:an amino acid mutation of Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; andan amino acid mutation of Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.

3. The coronavirus according to claim 1 wherein the replicase gene encodes a protein comprising the amino acid mutations Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7; Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.

4. The coronavirus according to claim 1 wherein the replicase gene encodes a protein comprising the amino acid mutations Pro to Leu at the position corresponding to position 85 of SEQ ID NO: 6; Val to Leu at the position corresponding to position 393 of SEQ ID NO: 7; Leu to Ile at the position corresponding to position 183 of SEQ ID NO: 8; and Val to Ile at the position corresponding to position 209 of SEQ ID NO: 9.

5. The coronavirus according to claim 1 wherein the replicase gene comprises at least one nucleotide substitutions selected from: compared to the sequence shown as SEQ ID NO: 1.C to Tat nucleotide position 12137; andG to C at nucleotide position 18114;compared to the sequence shown as SEQ ID NO: 1;and optionally, comprises one or more nucleotide substitutions selected from T to A at nucleotide position 19047; andG to A at nucleotide position 20139;

6. The coronavirus according to claim 1 which is an infectious bronchitis virus (IBV).

7. The coronavirus according to claim 1 which is IBV M41.

8. The coronavirus according to claim 7, which comprises an S protein at least, part of which is from an IBV serotype other than M41.

9. The coronavirus according to claim 8, wherein the S1 subunit is from an IBV serotype other than M41.

10. The coronavirus according to claim 8, wherein the S protein is from an IBV serotype other than M41.

11. The coronavirus according to claim 1 which has reduced pathogenicity compared to a coronavirus expressing a corresponding wild-type replicase, wherein the virus is capable of replicating without being pathogenic to the embryo when administered to an embryonated egg.

12. A variant replicase gene as defined in claim 1.

13. A protein encoded by a variant coronavirus replicase gene according to claim 12.

14. A plasmid comprising a replicase gene according to claim 12.

15. A method for making the coronavirus according to claim 1 which comprises the following steps:(i) transfecting a plasmid according to claim 14 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.

16. The method according to claim 15, wherein the recombining virus is a vaccinia virus.

17. The method according to claim 15 which also includes the step:(v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).

18. A cell capable of producing a coronavirus according to claim 1.

19. A vaccine comprising a coronavirus according to claim 1 and a pharmaceutically acceptable carrier.

20. A method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to claim 19 to the subject.

21. The method of claim 20, wherein the disease is infectious bronchitis (IB).

22. The method according to claim 20 wherein the method of administration is selected from the group consisting of; eye drop administration, intranasal administration, drinking water administration, post-hatch injection and in ovo injection.

23. The method according to claim 21 wherein the administration is in ovo vaccination.

24. A method for producing a vaccine according to claim 19, which comprises the step of infecting a cell according to claim 18 with a coronavirus according to claim 1.

25. The coronavirus according to claim 1, further comprising a mutation in one or both of nsp-15 and nsp-16.Referenced CitedU.S. Patent Documents

7452542 November 18, 2008 Denison

Foreign Patent Documents

WO-2004/092360 October 2004 WO
WO-2005/049814 June 2005 WO
WO-2007/078203 July 2007 WO
WO-2011/004146 January 2011 WO

Other references

  • Sperry Journal of Virology, 2005, vol. 79, No. 6, pp. 3391-3400.
  • Altschul et al., Basic local alignment search tool. J. Mol. Biol. 215: 403-10 (1990).
  • Ammayapppan et al., Identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain Arkansas DPI, Arch. Virol., 154(3):495-9 (2009).
  • Anonymous: “EM_STD:KF377577”, Oct. 30, 2013.
  • Armesto et al., A recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype, PLoS One, 6(8):e24352 (2011).
  • Armesto et al., The replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity, PLoS One, 4(10):e7384 (2009).
  • Armesto et al., Transient dominant selection for the modification and generation of recombinant infectious bronchitis coronaviruses, Methods Mol. Biol., 454:255-73 (2008).
  • Ausubel et al., Short Protocols in Molecular Biology, 4th edition, Chapter 18 (1999).
  • Britton et al., Generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection, J. Virol. Methods, 123(2):203-11 (2005).
  • Britton et al., Modification of the avian coronavirus infectious bronchitis virus for vaccine development, Bioeng. Bugs., 3(2):114-9 (2012).
  • Casais et al., Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism, J. Virol., 77(16):9084-9 (2003).
  • Casais et al., Reverse genetics system for the avian coronavirus infectious bronchitis virus, J. Virol., 75(24):12359-69 (2001).
  • Devereux et al., A comprehensive set of sequence analysis programms for the VAX. Nucl. Acids Res.12: 387-95 (1984).
  • Cavanagh et al., Manipulation of the infectious bronchitis coronavirus genome for vaccine development and analysis of the accessory proteins, Vaccine, 25(30):5558-62 (2007).
  • International Preliminary Report on Patentability, International Application No. PCT/GB2015/052124, dated Jan. 24, 2017.
  • International Search Report and Written Opinion, International Application No. PCT/GB2015/052124, dated Oct. 9, 2015.
  • Larkin et al., Clustal W and Clustal X version 2.0, Bioinformatics, 23(21):2947-8 (2007).
  • Menachery et al., Attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2′-o-methyltransferase activity, J. Virol., 88(8):4251-64 (2014).
  • Tatusova et al., BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences, FEMS Microbiol. Lett., 174(2):247-50 (1999).
  • Wang et al., Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence, Virology, 371(2):418-29 (2008).
  • Wei et al., Development and characterization of a recombinant infectious bronchitis virus expressing the ectodomain region of S1 gene of H120 strain, Appl. Microbiol. Biotechnol., 98(4):1727-35 (2014).

Patent HistoryPatent number: 10130701
Type: Grant
Filed: Jul 23, 2015
Date of Patent: Nov 20, 2018
Patent Publication Number20170216427
AssigneeTHE PIRBRIGHT INSTITUTE (Woking, Pirbright)
InventorsErica Bickerton (Woking), Sarah Keep (Woking), Paul Britton (Woking)
Primary ExaminerBao Q Li
Application Number: 15/328,179
ClassificationsCurrent U.S. ClassCoronaviridae (e.g., Neonatal Calf Diarrhea Virus, Feline Infectious Peritonitis Virus, Canine Coronavirus, Etc.) (424/221.1)
International Classification: A61K 39/215 (20060101); C12N 7/00 (20060101); C12N 9/12 (20060101); A61K 39/00 (20060101);
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NYC Mayor De Blasio tells citizens: We own your bodies, and we can force you to be injected with anything we want

Friday, April 12, 2019 by: Mike Adams

(Natural News) NYC Mayor Bill de Blasio has declared that residents do not own their own bodies. The city of New York can demand that all citizens be injected with literally anything the government declares to be a “vaccine,” even when those vaccines contain aborted human fetal tissue cells, toxic aluminum metals, inflammatory adjuvants and other dangerous, deadly chemicals.

This is the latest attempt by authorities in New York to obliterate human rights and roll out a medical dictatorship where citizens have zero rights to defend their own bodies against risky medial interventions that are demanded at gunpoint.

PJ Media, which has emerged as one of the best independent media websites covering liberty and individual rights, has published an especially noteworthy article on this issue. Authored by Megan Fox, the story is entitled, “Mayor Bill de Blasio’s Mandatory Measles Vaccination Order Faces Legal Challenges.”

We are republishing it here with full credit to the original author and PJ Media website. This in important read. Share everywhere.

Mayor Bill de Blasio’s Mandatory Measles Vaccination Order Faces Legal Challenges

by Megan Fox, PJMedia.com

In an unusual and extreme move, New York Mayor Bill de Blasio declared a state of emergency over a measles outbreak in the Orthodox Jewish community and is demanding forced vaccinations of everyone within four zip codes of the affected areas. Violators face fines up to $1000. This includes babies six months of age, even though the MMR is not recommended for anyone under twelve months of age.

The Children’s Health Defense will be filing a legal challenge to the order, which comes on the heels of a New York Supreme Court ruling that struck down the Rockland County banon unvaccinated children in public spaces.

Children’s Health Defense (CHD) is supporting a legal challenge to this dangerous, unprecedented overreach. While the City has unquestionable authority to control disease outbreaks, it may not violate the bedrock principle of prior, free and informed consent to all medical interventions, including vaccines. This is a fundamental human right. The City may quarantine, isolate, trace contacts and strongly urge vaccination, but it may not impose such a draconian mandate without demonstrating necessity, reasonableness, proportionality, harm avoidance, non-discrimination, due process and equal protection. The Commissioner has failed to do this; the City’s actions violate New York State law.

CHD board member Mary Holland commented, “I am shocked that Mayor de Blasio would resort to such police state techniques to control an outbreak of measles. I don’t believe the City’s actions will withstand legal scrutiny.” CHD Chairman Robert F. Kennedy Jr. is confident their legal challenge will prevail.

This case goes beyond a dispute over religious freedom. Thanks to the Merck federal whistleblower litigation, we now know that Merck’s MMR should have never been approved, much less mandated. To get its license Merck allegedly ordered its scientists to falsify efficacy data to fraudulently conceal the fact that the mumps component quickly wanes, triggering dangerous outbreaks in older populations where it can cause sterility in men and women. The Centers for Disease Control and Prevention (CDC) reported 150 outbreaks resulting in 9,200 cases of mumps in fully vaccinated adults, dwarfing the recent measles outbreaks. We are confident that no American court will allow government bureaucrats to force American citizens to take risky pharmaceutical products against their will.

Merck is currently defending itself against claims of falsifying data brought by two former employees.

Medical corporation Merck & Co. will decidedly face the music in the ongoing class action and related anti-trust lawsuit involving its mumps vaccine – a product routinely given to babies and children for generations. The issue, which involves allegations of false compliance with FDA standards for vaccines, prompted a False Claims Act lawsuit: United States v. Merck & Co. This case was commenced by two virologists once employed with Merck, alleges a systematic and long-standing commitment by the company to lying about the efficacy of its mumps vaccination, thereby prompting possible exposure to liability under the federal False Claims Act.

Governor Cuomo voiced concerns about the legality of de Blasio’s emergency order to forcibly vaccinate conscientious objectors. “Look, it’s a serious public health concern, but it’s also a serious First Amendment issue and it is going to be a constitutional, legal question,” Cuomo said in a radio interview on WAMC. “Do we have the right — does society, government have the right to say ‘you must vaccinate your child because I’m afraid your child is going to infect my child, even if you don’t want it done and even if it violates your religious beliefs?”

Some have asked how de Blasio is planning to determine who is or isn’t vaccinated to enforce his order. According to the mayor, they will be using “disease detectives.” de Blasio explained, “It parallels what a police detective does. If someone has symptoms, they will literally interview them to figure out everywhere they’ve been, everyone they might have come in contact with, and then they go reach out to that whole network to make sure people are vaccinated.” It’s unclear whether “make sure people are vaccinated” means “hold them down and inject them against their will.” (Click to Source)

Read more stories on liberty and individual rights at PJmedia.com. Stay informed about vaccine dangers and vaccine industry propaganda by reading Vaccines.news.

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Same corrupt California government that pushed vaccine mandates now forming a “fake news” authority to BLOCK news sources they don’t like… California to become Communist China

06/28/2018 / By Ethan Huff

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Liberals in the Golden State have come up with yet another way to expand authoritarian control over the people who live there. It’s known as Senate Bill 1424, and it would basically eliminate all independent press in California, dubbing any information that establishment politicians disagree with as “fake news.”

If it ends up passing and being signed into law, SB 1424 will mandate that the California Attorney General create an advisory committee by April 1, 2019, to police the information that’s spread via social media and other platforms to determine whether or not it’s “true.” The state’s ministry of propaganda will then come up with a plan of attack to eliminate anything that’s deemed “false.”

Since “fake news” is a subjective term that varies in its substance depending on who you ask, there’s no real way to police it. But California politicians sure are going to try, and what it will mean (based on the vast majority of their political leanings) is the silencing of all dissenting viewpoints that don’t fit whatever official narrative the government is trying to push.

The Electronic Frontier Foundation (EFF), a freedom advocacy group, is vehemently opposed to the bill, having called it both “flawed” and “misguided.” Because there are already laws on the books that hold news outlets accountable whenever they print defamatory material, there’s simply no need to establish a ministry of “truth” that’s tasked with authoritatively declaring what information does and does not qualify as such.

“The group argues the measure would make the government and advisory group responsible for deciding what is true or false,” explains EFF about its position. “It also points out the First Amendment prevents content-based restrictions, even if the statements of ‘admittedly false.’”

Another California bill, AB 155, is attempting to force public school teachers to indoctrinate students about “fake news”

This is hardly the first egregiously unconstitutional legislative bill to come down the pipeline in California. Many Natural News readers will recall the infamous Senate Bill 277, the legislative baby of corrupt Big Pharma shill Richard Pan, that eliminated all philosophical and religious exemptions for vaccines in the state of California.

There’s also AB 155, a newly proposed bill that would require public school teachers to indoctrinate their students about the concept of “fake news” – presumably framing it in terms of President Trump’s alleged “Russian meddling” in the 2016 presidential election that led to the spread of what liberals claim was “fake news” all over social media. And who could forget SB 48, yet another California masterpiece that requires public school children to be taught that transgenderism is completely normal and natural.

It’s all part of the lunatic left’s insidious plan to transform far-left states like California into certifiably Communist police states that control pretty much everything that people learn, eat, say, and inject or put inside their bodies. Freedom of choice, in other words, is on the verge of completely disappearing from the Left Coast in this regard, and all in the name of political correctness and “tolerance.”

Soon Californians won’t even be able to speak freely without facing some sort of penalty for the “crime” of saying the wrong thing. This is actually already in the works with regards to LGBT issues and sexual orientation with the introduction of AB 2943. As we recently reported, the threatening legislation would make it a crime for licensed psychologists and counselors to offer reparative or “conversion” therapy to willing individuals struggling with gender identity issues.

Political correctness will be the death of this nation, and certainly of freedom of speech. To keep up with the latest news, visit ThoughtPolice.news.  (Click to Source)

Sources for this article include:

DailyWire.com

NaturalNews.com

NaturalNews.com

 
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Kidney doctor who sounded the alarm on vaccine damage was targeted for murder when the brake lines of her vehicle were secretly clamped

suzanne-humphries

(Natural News) Many in the natural health community are already aware of the recent death threat that vaccine truth advocate Dr. Suzanne Humphries received from an anonymous assailant. But what you may not realize is that Dr. Humphries was previously targeted for murder when someone maliciously severed the brake lines on her vehicle.

During a recent interview with Mike Adams, the Health Ranger, Dr. Humphries bore it all about her history working in medicine; the criticism and animosity she’s faced over the years for departing from its dogma; and more recently the vulgar threat she received on her life via anonymous email. Dr. Humphries also told of how her work in exposing vaccine truth hasn’t exactly done her any favors in terms of her personal safety.

It was right around the time when Dr. Humphries began to notice, and talk about, the increasing number of kidney conditions she was observing in conjunction with influenza vaccination that she quickly landed in the crosshairs of pro-vaccine cultists. She was falsely accused of being an “anti-vaxxer,” a categorical branding that represents a type of medical scarlet letter, and one that draws plenty of ire and hate from the more extremist “jabbers” who believe that people who don’t vaccinate put all of society at risk.

If a person really believes this unsubstantiated dogma, it’s easy to see how angered they might become at people like Dr. Humphries who are spreading a different narrative. To pro-vaccination cultists like the anonymous assailant who most recently threatened Dr. Humphries’s life, warning people about the dangers of vaccines basically makes one an infidel who must be taken out for the protection of the “herd.”

“The brake lines to my Honda CR-V were clamped, and there was something wrong with how the car was functioning,” Dr. Humphries revealed for the first time, recalling an incident that’s she never gone public with that occurred back around 2010. “It was immediately taken into the service station, and the service station attendant was practically in tears. He said, ‘This was a malicious act.’”

The full video of Dr. Humphries’s exclusive interview with the Health Ranger is available on YouTube and on Vimeo.

Dr. Humphries says she’s also been targeted with a crossbow arrow on her lawn, aggressor who turned gas line on inside her home

Dr. Humphries also recalled at least three other incidents besides the recent death threat that she’s faced over the years. One of them was a large crossbow arrow that someone had stuck into the ground on her front lawn. Another was an incident where someone had broken into her home and turned on the gas line, presumably to kill whoever might be inside with carbon monoxide.

On a fourth occasion, Dr. Humphries’s dog was sprayed with some kind of chemical that left him blind for the night (but he apparently later recovered). Dr. Humphries is sure that a skunk wasn’t the culprit, and believes that someone may have targeted her pup with pepper spray for malicious, threatening purposes.

When Dr. Humphries reported these various incidents to the police, she was told that there was nothing law enforcement could do other than to simply hold onto the records in case more happened later – which is completely illogical. They never even attempted to conduct an investigation, and now Dr. Humphries is facing yet another, more serious, threat that law enforcement similarly wants to ignore. Dr. Humphries has also received countless nasty emails and other threats against her, none of which have been taken seriously by law enforcement.

“What I’m learning is that the so-called ‘protective authorities’ are not actually that,” Dr. Humphries soberly admitted to the Health Ranger. (Click to Source)

MASSIVE FLU OUTBREAK? HERE’S THE REAL STORY THE MEDIA WON’T TOUCH.

The lies, the hoax, the scandal

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In case you haven’t been following the uproar over the flu outbreak, you’ve missed the fact that…

Health authorities admit this year’s flu vaccine is only 10% effective.

But of course, they urge you to take the vaccine anyway.

Why is this year’s vaccine ineffective? Because it’s made using chicken eggs, and researchers have discovered that the flu virus—which is placed in the vaccine—mutates in chicken eggs.

Therefore, by the time a person takes the flu shot, he’s not being protected against this year’s seasonal flu virus. He’s being protected against a mutated virus that isn’t causing the flu this year.

This is the conventional explanation. If you think it’s the whole story, I have condos for sale on the moon.

You see, vaccines have been made using chicken eggs—not just this year—but for the past 70 years.

Oops.

That would mean the flu vaccine has been ineffective for decades.

Healthline.com: “The majority of flu vaccines are grown in chicken eggs, a method of vaccine development that’s been used for 70 years.”

But wait. There’s more. Much more. I’ll break it down into several scandals.

SCANDAL ONE: (I covered this the other day): Dr. Peter Doshi, writing in the online BMJ (British Medical Journal), reveals a monstrosity.

As Doshi states, every year, hundreds of thousands of respiratory samples are taken from flu patients in the US and tested in labs. Here is the kicker: only a small percentage of these samples show the presence of a flu virus.

This means: most of the people in America who are diagnosed by doctors with the flu have no flu virus in their bodies.

So they don’t have the flu.

Therefore, even if you assume the flu vaccine is useful and safe, it couldn’t possibly prevent all those “flu cases” that aren’t flu cases.

The vaccine couldn’t possibly work.

The vaccine isn’t designed to prevent fake flu, unless pigs can fly.

Here’s the exact quote from Peter Doshi’s BMJ review, “Influenza: marketing vaccines by marketing disease” (BMJ 2013; 346:f3037):

“…even the ideal influenza vaccine, matched perfectly to circulating strains of wild influenza and capable of stopping all influenza viruses, can only deal with a small part of the ‘flu’ problem because most ‘flu’ appears to have nothing to do with influenza. Every year, hundreds of thousands of respiratory specimens are tested across the US. Of those tested, on average 16% are found to be influenza positive.”

“…It’s no wonder so many people feel that ‘flu shots’ don’t work: for most flus, they can’t.”

Because most diagnosed cases of the flu aren’t the flu.

So even if you’re a true believer in mainstream vaccine theory, you’re on the short end of the stick here. They’re conning your socks off.

The basic flu symptoms—cough, fever, chills, sore throat, muscle aches, weakness—can be caused by a variety of factors that have nothing to do with a flu virus.

SCANDAL TWO: In December of 2005, the British Medical Journal (online) published another shocking Peter Doshi report, which created tremors through the halls of the Centers for Disease Control (CDC), where “the experts” used to tell the press that 36,000 people in the US die every year from the flu.

Here is a quote from Doshi’s report, “Are US flu death figures more PR than science?”(BMJ 2005; 331:1412):

“[According to CDC statistics], ‘influenza and pneumonia’ took 62,034 lives in 2001—61,777 of which were attributable to pneumonia and 257 to flu, and in only 18 cases was the flu virus positively identified.”

Boom.

You see, the CDC has created one overall category that combines both flu and pneumonia deaths. Why do they do this? Because they disingenuously assume that the pneumonia deaths are complications stemming from the flu.

This is an absurd assumption. Pneumonia has a number of causes.

But even worse, in all the flu and pneumonia deaths, only 18 revealed the presence of an influenza virus.

Therefore, the CDC could not say, with assurance, that more than 18 people died of influenza in 2001. Not 36,000 deaths. 18 deaths.

Doshi continued his assessment of published CDC flu-death statistics: “Between 1979 and 2001, [CDC] data show an average of 1348 [flu] deaths per year (range 257 to 3006).” These figures refer to flu separated out from pneumonia.

This death toll is obviously far lower than the old parroted 36,000 figure.

However, when you add the sensible condition that lab tests have to actually find the flu virus in patients, the numbers of flu deaths plummet even further.

In other words, it’s all promotion and hype.

“Well, uh, we used to say that 36,000 people die from the flu every year in the US. But actually, all we can prove is about 20 deaths. However, we can’t admit that, because if we did, we’d be exposing our gigantic psyop. The whole campaign to scare people into getting a flu shot would have about the same effect as warning people to carry iron umbrellas, in case toasters fall out of upper-story windows…and, by the way, we’d be put in prison for fraud.”

SCANDAL THREE: The so-called Swine Flu pandemic of 2009. This one is a real eye-opener. The CDC was caught with its pants down.

Swine Flu was hyped to the sky by the CDC. The Agency was calling for all Americans to take the Swine Flu vaccine. Remember?

The problem was, the CDC was concealing a very dirty secret.

At the time, star CBS investigative reporter, Sharyl Attkisson, was working on the Swine Flu story. She discovered that the CDC had secretly stopped counting cases of the illness—while, of course, continuing to warn Americans about its unchecked spread.

Understand that the CDC’s main job is counting cases and reporting the numbers.

What was the Agency up to?

Here is an excerpt from my 2014 interview with Sharyl Attkisson:

Rappoport: In 2009, you spearheaded coverage of the so-called Swine Flu pandemic. You discovered that, in the summer of 2009, the Centers for Disease Control, ignoring their federal mandate, [secretly] stopped counting Swine Flu cases in America. Yet they continued to stir up fear about the “pandemic,” without having any real measure of its impact. Wasn’t that another investigation of yours that was shut down? Wasn’t there more to find out?

Attkisson: The implications of the story were even worse than that. We discovered through our FOI efforts that before the CDC mysteriously stopped counting Swine Flu cases, they had learned that almost none of the cases they had counted as Swine Flu was, in fact, Swine Flu or any sort of flu at all! The interest in the story from one [CBS] executive was very enthusiastic. He said it was “the most original story” he’d seen on the whole Swine Flu epidemic. But others pushed to stop it [after it was published on the CBS News website] and, in the end, no [CBS television news] broadcast wanted to touch it. We aired numerous stories pumping up the idea of an epidemic, but not the one that would shed original, new light on all the hype. It was fair, accurate, legally approved and a heck of a story. With the CDC keeping the true Swine Flu stats secret, it meant that many in the public took and gave their children an experimental vaccine that may not have been necessary.

—end of interview excerpt—

I’ll add a few details. It was routine for doctors all over America to send blood samples from patients they’d diagnosed with Swine Flu, or the “most likely” Swine Flu patients, to labs for testing. And overwhelmingly, those samples were coming back with the result: not Swine Flu, not any kind of flu.

That was the big secret. That’s what the CDC was hiding. That’s why they stopped reporting Swine Flu case numbers. That’s what Attkisson had discovered. That’s why she was shut down.

But it gets even worse.

Because about three weeks after Attkisson’s findings were published on the CBS News website, the CDC, obviously in a panic, decided to double down. If one lie is exposed, tell an even bigger one. A much bigger one.

Here, from a November 12, 2009, WebMD article is the CDC’s response: “Shockingly, 14 million to 34 million U.S. residents — the CDC’s best guess is 22 million — came down with H1N1 swine flu by Oct. 17 [2009].” (“22 million cases of Swine Flu in US,” by Daniel J. DeNoon).

Are your eyeballs popping? They should be.

In the summer of 2009, the CDC secretly stops counting Swine Flu cases in America, because the overwhelming percentage of lab tests from likely Swine Flu patients shows no sign of Swine Flu or any other kind of flu.

There is no Swine Flu epidemic.

Then, the CDC estimates there are 22 MILLION cases of Swine Flu in the US.

So now…when health officials begin waving red flags and raising alarms about a current viral flu outbreak, it would be more than reasonable to demand they answer questions about their past lies and deceptions.

Unless you just want to take them at their word.

If so, good luck. (Click to Source)

This article first appeared at NoMoreFakeNews.com.