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Creator Of US BioWeapons Act Says Coronavirus Is Biological Warfare Weapon

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Creator Of US BioWeapons Act Says Coronavirus Is Biological Warfare Weapon

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Mon, 02/03/2020 - 17:25
 

Via GreatGameIndia.com,

In an explosive interview Dr. Francis Boyle, who drafted the Biological Weapons Act has given a detailed statement admitting that the 2019 Wuhan Coronavirus is an offensive Biological Warfare Weapon and that the World Health Organization (WHO) already knows about it.

 

Dr-Francis-Boyle-Coronavirus-Biological-

Dr. Francis Boyle Creator Of BioWeapons Act Says Coronavirus Is Biological Warfare Weapon

 
 

Francis Boyle is a professor of international law at the University of Illinois College of Law. He drafted the U.S. domestic implementing legislation for the Biological Weapons Convention, known as the Biological Weapons Anti-Terrorism Act of 1989, that was approved unanimously by both Houses of the U.S. Congress and signed into law by President George H.W. Bush.

In an exclusive interview given to Geopolitics and Empire, Dr. Boyle discusses the coronavirus outbreak in Wuhan, China and the Biosafety Level 4 laboratory (BSL-4) from which he believes the infectious disease escaped. He believes the virus is potentially lethal and an offensive biological warfare weapon or dual-use biowarfare weapons agent genetically modified with gain of function properties, which is why the Chinese government originally tried to cover it up and is now taking drastic measures to contain it.

The Wuhan BSL-4 lab is also a specially designated World Health Organization (WHO) research lab and Dr. Boyle contends that the WHO knows full well what is occurring.

Dr. Boyle also touches upon GreatGameIndia‘s exclusive report Coronavirus Bioweapon – where we reported in detail how Chinese Biowarfare agents working at the Canadian lab in Winnipeg were involved in the smuggling of Coronavirus to Wuhan’s lab from where it is believed to have been leaked.

Watch Dr. Francis Boyle’s interview with Geopolitics and Empire below:

Dr. Boyle’s position is in stark contrast to the mainstream media’s narrative of the virus being originated from the seafood market, which is increasingly being questioned by many experts.

Recently, American Senator Tom Cotton of Arkansas also dismantled the mainstream media’s claim on Thursday that pinned the coronavirus outbreak on a market selling dead and live animals.

In a video accompanying his post, Cotton explained that the Wuhan wet market (which Cotton incorrectly referred to as a seafood market) has been shown by experts to not be the source of the deadly contagion.

Cotton referenced a Lancet study which showed that many of the first cases of the novel coronavirus, including patient zero, had no connection to the wet market — devastatingly undermining mainstream media’s claim.

“As one epidemiologist said: ‘That virus went into the seafood market before it came out of the seafood market.’ We still don’t know where it originated,” Cotton said.

“I would note that Wuhan also has China’s only bio-safety level four super laboratory that works with the world’s most deadly pathogens to include, yes, coronavirus.”

Such concerns have also been raised by J.R. Nyquist, the well known author of the books “Origins of the Fourth World War” and “The Fool and His Enemy,” as well as co-author of “The New Tactics of Global War”. In his insightful article he published secret speechs given to high-level Communist Party cadres by Chinese Defense Minister Gen. Chi Haotian explaining a long-range plan for ensuring a Chinese national renaissance – the catalyst for which would be China’s secret plan to weaponiz viruses.

Nyquist gave three different data points for making his case in analyzing Coronavirus. He writes:

The third data point worth considering: the journal GreatGameIndia has published a piece titled “Coronavirus Bioweapon – How China Stole Coronavirus From Canada And Weaponized It.”

The authors were clever enough to put Khan’s Virology Journal article together with news of a security breach by Chinese nationals at the Canadian (P4) National Microbiology Lab in Winnipeg, where the novel coronavirus was allegedly stored with other lethal organisms. Last May, the Royal Canadian Mounted Police were called in to investigate; by late July the Chinese were kicked out of the facility. The chief Chinese scientist (Dr. Xiangguo Qiu) was allegedly making trips between Winnipeg and Wuhan.

Here we have a plausible theory of the NCoV organism’s travels: first discovered in Saudi Arabia, then studied in Canada from whence it was stolen by a Chinese scientist and brought to Wuhan. Like the statement of Taiwan’s intelligence chief in 2008, the GreatGameIndia story has come under intensive attack. Whatever the truth, the fact of proximity and the unlikelihood of mutation must figure into our calculations.

It’s highly probable that the 2019-nCoV organism is a weaponized version of the NCoV discovered by Saudi doctors in 2012.

Meanwhile, the mainstream media’s narrative still maintains that the origin of the 2019 Coronavirus is the Wuhan Seafood Market. After GreatGameIndia published the story on Coronavirus Bioweapon – not only were our databse tinkered with and our reports blocked by Facebook on the flimsy reason that they could not find GreatGameIndia Facebook page, but the report itself was viciously attacked by Foreign Policy magazine, PolitiFact (known widely as Facebook’s propaganda arm) and BuzzFeedNews.

https://www.zerohedge.com/health/creator-bioweapons-act-says-coronavirus-biological-warfare-weapon

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It is not GreatGameIndia alone which is being viciously attacked. Zero Hedge, a popular alternate media blog was suspended by Twitter for publishing a story related to a study by Indian scientists finding 2019 Wuhan Coronavirus to be not naturally evolved, raising the possibility of it being created in a lab. Shockingly, the study itself came under intense online criticism by Social Media experts resulting in the scientists withdrawing the paper.

In retaliation India has launched a full-scale investigation against China’s Wuhan Institute of Virology. The Indian government has ordered an inquiry into a study conducted in the Northeastern state of Nagaland (close to China) by researchers from the U.S., China and India on bats and humans carrying antibodies to deadly viruses like Ebola.

The study came under the scanner as two of the 12 researchers belonged to the Wuhan Institute of Virology’s Department of Emerging Infectious Diseases, and it was funded by the United States Department of Defense’s Defense Threat Reduction Agency (DTRA).

The study, conducted by scientists of the Tata Institute of Fundamental Research, the National Centre for Biological Sciences (NCBS), the Wuhan Institute of Virology, the Uniformed Services University of the Health Sciences in the U.S. and the Duke-National University in Singapore, is now being investigated for how the scientists were allowed to access live samples of bats and bat hunters (humans) without due permissions.

The results of the study were published in October last year in the PLOS Neglected Tropical Diseases journal, originally established by the Bill and Melinda Gates Foundation.

As the author J.R. Nyquist puts it:

We must have an investigation of the outbreak in Wuhan. The Chinese must grant the world total transparency. The truth must come out. If Chinese officials are innocent, they have nothing to hide. If they are guilty, they will refuse to cooperate.

The real concern here is whether the rest of the world has the courage to demand a real and thorough investigation. We need to be fearless in this demand and not allow “economic interests” to play a coy and dishonest game of denial. We need an honest inquiry. We need it now.

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Another thread here is worth a look.....Media and the establishment are trying to discount the patent information.......which is public record........at the same time the coronavirus was shipped to Canada for research......and somehow made it to China....and who knows what enhancements/changes were made there.......read the thread......and draw your own conclusions.....       CL

 

 

 

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I watched this video last night with my mouth wide open in the shock that this could actually be a possibility.  China has stolen intelectual property for years but if they actually stole this virus from Canada and somehow accidently leaked it from a WHO BSL-4 FACILITY in Wuhan, then tried to cover it up is unforgivable!

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Dr. Francis Boyle Creator Of BioWeapons Act Says Coronavirus Is Biological Warfare Weapon
 

 

SOURCE: GREAT GAME INDIA In an explosive interview Dr. Francis Boyle, who drafted the Biological Weapons Act has given a detailed statement admitting that the 2019 Wuhan Coronavirus is an offensive Biological Warfare Weapon and that the World Health Organization (WHO) already knows about it. Francis Boyle is a professor of international law at the University of Illinois College of Law. He drafted the U.S. domestic implementing legislation for the Biological Weapons Convention, known as the Biological Weapons Anti-Terrorism Act of 1989, that was approved unanimously by both Houses of the U.S. Congress and signed into law by President George H.W. Bush. In an exclusive interview given to Geopolitics and Empire, Dr. Boyle discusses the coronavirus outbreak in Wuhan, China and the Biosafety Level 4 laboratory (BSL-4) from which he believes the infectious disease escaped. He believes the virus is potentially lethal and an offensive biological warfare weapon or dual-use biowarfare weapons agent genetically modified with gain of function properties, which is why the Chinese government originally tried to cover it up and is now taking drastic measures to contain it. The Wuhan BSL-4 lab is also a specially designated World Health Organization (WHO) research lab and Dr. Boyle contends that the WHO knows full well what is occurring. Dr. Boyle also touches upon GreatGameIndia‘s exclusive report Coronavirus Bioweapon – where we reported in detail how Chinese Biowarfare agents working at the Canadian lab in Winnipeg were involved in the smuggling of Coronavirus to Wuhan’s lab from where it is believed to have been leaked. Dr. Boyle’s position is in stark contrast to the mainstream media’s narrative of the virus being originated from the seafood market, which is increasingly being questioned by many experts. Recently, American Senator Tom Cotton of Arkansas also dismantled the mainstream media’s claim on Thursday that pinned the coronavirus outbreak on a market selling dead and live animals. In a video accompanying his post, Cotton explained that the Wuhan wet market (which Cotton incorrectly referred to as a seafood market) has been shown by experts to not be the source of the deadly contagion. Cotton referenced a Lancet study which showed that many of the first cases of the novel coronavirus, including patient zero, had no connection to the wet market — devastatingly undermining mainstream media’s claim. “As one epidemiologist said: ‘That virus went into the seafood market before it came out of the seafood market.’ We still don’t know where it originated,” Cotton said. “I would note that Wuhan also has China’s only bio-safety level four super laboratory that works with the world’s most deadly pathogens to include, yes, coronavirus.” Such concerns have also been raised by J.R. Nyquist, the well known author of the books “Origins of the Fourth World War” and “The Fool and His Enemy,” as well as co-author of “The New Tactics of Global War”. In his insightful article he published secret speechs given to high-level Communist Party cadres by Chinese Defense Minister Gen. Chi Haotian explaining a long-range plan for ensuring a Chinese national renaissance – the catalyst for which would be China’s secret plan to weaponiz viruses. Nyquist gave three different data points for making his case in analyzing Coronavirus. He writes: The third data point worth considering: the journal GreatGameIndia has published a piece titled “Coronavirus Bioweapon – How China Stole Coronavirus From Canada And Weaponized It.” The authors were clever enough to put Khan’s Virology Journal article together with news of a security breach by Chinese nationals at the Canadian (P4) National Microbiology Lab in Winnipeg, where the novel coronavirus was allegedly stored with other lethal organisms. Last May, the Royal Canadian Mounted Police were called in to investigate; by late July the Chinese were kicked out of the facility. The chief Chinese scientist (Dr. Xiangguo Qiu) was allegedly making trips between Winnipeg and Wuhan. Here we have a plausible theory of the NCoV organism’s travels: first discovered in Saudi Arabia, then studied in Canada from whence it was stolen by a Chinese scientist and brought to Wuhan. Like the statement of Taiwan’s intelligence chief in 2008, the GreatGameIndia story has come under intensive attack. Whatever the truth, the fact of proximity and the unlikelihood of mutation must figure into our calculations. It’s highly probable that the 2019-nCoV organism is a weaponized version of the NCoV discovered by Saudi doctors in 2012. Meanwhile, the mainstream media’s narrative still maintains that the origin of the 2019 Coronavirus is the Wuhan Seafood Market. After GreatGameIndia published the story on Coronavirus Bioweapon – not only were our databse tinkered with and our reports blocked by Facebook on the flimsy reason that they could not find GreatGameIndia Facebook page, but the report itself was viciously attacked by Foreign Policy magazine, PolitiFact (known widely as Facebook’s propaganda arm) and BuzzFeedNews. It is not GreatGameIndia alone which is being viciously attacked. Zero Hedge, a popular alternate media blog was suspended by Twitter for publishing a story related to a study by Indian scientists finding 2019 Wuhan Coronavirus to be not naturally evolved, raising the possibility of it being created in a lab. Shockingly, the study itself came under intense online criticism by Social Media experts resulting in the scientists withdrawing the paper.

idrw.org .Read more at India No 1 Defence News Website https://idrw.org/dr-francis-boyle-creator-of-bioweapons-act-says-coronavirus-is-biological-warfare-weapon/ .

https://idrw.org/dr-francis-boyle-creator-of-bioweapons-act-says-coronavirus-is-biological-warfare-weapon/

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Coronavirus may have originated in lab linked to China's biowarfare program

 
Hospital staff wash the emergency entrance of Wuhan Medical Treatment Center, where some infected with a new virus are being treated, in Wuhan, China, Wednesday, Jan. 22, 2020. The number of cases of a new coronavirus from Wuhan has risen to over 400 in China health authorities said Wednesday. (AP Photo/Dake Kang)
Hospital staff wash the emergency entrance of Wuhan Medical Treatment Center, where some infected with a new virus are being treated, in Wuhan, China, Wednesday, Jan. 22, 2020. The number of cases of a new coronavirus from Wuhan has risen ... more >
 
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By Bill Gertz - The Washington Times - Sunday, January 26, 2020

The deadly animal-borne coronavirus spreading globally may have originated in a laboratory in the city of Wuhan linked to China’s covert biological weapons program, said an Israeli biological warfare analyst.

Radio Free Asia last week rebroadcast a Wuhan television report from 2015 showing China’s most advanced virus research laboratory, known the Wuhan Institute of Virology. The laboratory is the only declared site in China capable of working with deadly viruses.

Dany Shoham, a former Israeli military intelligence officer who has studied Chinese biological warfare, said the institute is linked to Beijing’s covert bio-weapons program.

“Certain laboratories in the institute have probably been engaged, in terms of research and development, in Chinese [biological weapons], at least collaterally, yet not as a principal facility of the Chinese BW alignment,” Mr. Shoham told The Washington Times.

Work on biological weapons is conducted as part of dual civilian-military research and is “definitely covert,” he said in an email.



Mr. Shoham holds a doctorate in medical microbiology. From 1970 to 1991, he was a senior analyst with Israeli military intelligence for biological and chemical warfare in the Middle East and worldwide. He held the rank of lieutenant colonel.

China has denied having any offensive biological weapons, but a State Department report last year revealed suspicions of covert biological warfare work.

A Chinese Embassy spokesman did not return an email seeking comment.

Chinese authorities said they do not know the origin of the coronavirus, which has killed at least 80 and infected thousands.

Gao Fu, director of the Chinese Center for Disease Control and Prevention, told state-controlled media that initial signs indicated the virus originated from wild animals sold at a seafood market in Wuhan.

One ominous sign, said a U.S. official, is that false rumors circulating on the Chinese internet claim the virus is part of a U.S. conspiracy to spread germ weapons. That could indicate China is preparing propaganda outlets to counter any charges that the new coronavirus escaped from one of Wuhan’s civilian or defense research laboratories.

The World Health Organization is calling the microbe novel coronavirus 2019-nCoV. At a meeting Thursday in Geneva, the organization stopped short of declaring a public health emergency of international concern.

China has deployed military forces to Wuhan to halt all travel out of the city of 11 million people in an effort to contain the outbreak of the virus, which causes pneumonialike symptoms.

The Wuhan institute has studied coronaviruses including the strain that causes severe acute respiratory syndrome (SARS), H5N1 influenza virus, Japanese encephalitis and dengue. Researchers at the institute also have studied the germ that causes anthrax, a biological agent once developed in Russia.

“Coronaviruses [particularly SARS] have been studied in the institute and are probably held therein,” Mr. Shoham said. “SARS is included within the Chinese BW program, at large, and is dealt with in several pertinent facilities.”

It is not known whether the institute’s coronaviruses are specifically included in China’s biological weapons program but it is possible, he said.

Asked whether the new coronavirus may have leaked, Mr. Shoham said: “In principle, outward virus infiltration might take place either as leakage or as an indoor unnoticed infection of a person that normally went out of the concerned facility. This could have been the case with the Wuhan Institute of Virology, but so far there isn’t evidence or indication for such incident.”

After researchers sequence the genome of the new coronavirus, they might be able to determine or suggest its origin or source.

Biological weapons convention

Mr. Shoham, now with the Begin-Sadat Center for Strategic Studies at Bar Ilan University in Israel, said the virology institute is the only declared site in China known as P4 for pathogen level 4. That status indicates the institute uses the strictest safety standards to prevent the spread of the most dangerous and exotic microbes being studied.

The former Israeli military intelligence doctor also said suspicions were raised about the Wuhan Institute of Virology when a group of Chinese virologists working in Canada improperly sent to China samples of what he described as some of the deadliest viruses on earth, including the Ebola virus.

In a July article in the journal Institute for Defense Studies and Analyses, Mr. Shoham said the Wuhan institute was one of four Chinese laboratories engaged in some aspects of biological weapons development.

He said the secure Wuhan National Biosafety Laboratory at the institute was engaged in research on the Ebola, Nipah and Crimean-Congo hemorrhagic fever viruses.

The Wuhan virology institute is under the Chinese Academy of Sciences, but certain laboratories within it “have linkage with the PLA or BW-related elements within the Chinese defense establishment,” he said.

In 1993, China declared a second facility, the Wuhan Institute of Biological Products, as one of eight biological warfare research facilities covered by the Biological Weapons Convention, which China joined in 1985.

The Wuhan Institute of Biological Products is a civilian facility but is linked to the Chinese defense establishment. Mr. Shoham said it is thought to be involved in the Chinese Biological Weapons Convention program. China’s vaccine against SARS is probably produced there.

“This means the SARS virus is held and propagated there, but it is not a new coronavirus unless the wild type has been modified, which is not known and cannot be speculated at the moment,” he said.

The annual State Department report on arms treaty compliance stated last year that China engaged in activities that could support biological warfare.

“Information indicates that the People’s Republic of China engaged during the reporting period in biological activities with potential dual-use applications, which raises concerns regarding its compliance with the BWC,” said the report, adding that the United States suspects China failed to eliminate its biological warfare program as required by the treaty.

“The United States has compliance concerns with respect to Chinese military medical institutions’ toxin research and development because of the potential dual-use applications and their potential as a biological threat,” the report said.

The biosafety lab is about 20 miles from the Hunan Seafood Market, which reports from China say may have been the origin point of the virus.

Rutgers University microbiologist Richard Ebright told London’s Daily Mail that “at this point there’s no reason to harbor suspicions” that the lab may be linked to the virus outbreak.

https://www.washingtontimes.com/news/2020/jan/26/coronavirus-link-china-biowarfare-program-possible/

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Etc.)US Patent for Coronavirus Patent (Patent # 10,130,701)

 

 

US Patent 2015

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.

Skip to: Description  ·  Claims  ·  References Cited  · Patent History  ·  Patent History
 
=================
 
Justia  Patents  Coronaviridae (e.g., Neonatal Calf Diarrhea Virus, Feline Infectious Peritonitis Virus, Canine Coronavirus, Etc.)US Patent for Coronavirus Patent (Patent # 10,130,701)

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.

Skip to: Description  ·  Claims  ·  References Cited  · Patent History  ·  Patent History
Description
FIELD OF THE INVENTION

The present invention relates to an attenuated coronavirus comprising a variant replicase gene, which causes the virus to have reduced pathogenicity. The present invention also relates to the use of such a coronavirus in a vaccine to prevent and/or treat a disease.

BACKGROUND TO THE INVENTION

Avian infectious bronchitis virus (IBV), the aetiological agent of infectious bronchitis (IB), is a highly infectious and contagious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct. IBV is a member of the Order Nidovirales, Family Coronaviridae, Subfamily Corona virinae and Genus Gammacoronavirus; genetically very similar coronaviruses cause disease in turkeys, guinea fowl and pheasants.

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

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

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

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

It is important that new and safer vaccines are developed for the control of IBV. Thus there is a need for IBV vaccines which are not associated with these issues, in particular vaccines which may be used for in ovo vaccination.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have used a reverse genetics approach in order to rationally attenuate IBV. This approach is much more controllable than random attenuation following multiple passages in embryonated eggs because the position of each mutation is known and its effect on the virus, i.e. the reason for attenuation, can be derived.

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

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

The variant replicase gene may encode a protein comprising one or more amino acid mutations selected from the list of:

    • Pro to Leu at position 85 of SEQ ID NO: 6,
    • Val to Leu at position 393 of SEQ ID NO: 7;
    • Leu to Ile at position 183 of SEQ ID NO: 8;
    • Val to Ile at position 209 of SEQ ID NO: 9.

 

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

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

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

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

C to T at nucleotide position 12137;

G to C at nucleotide position 18114;

T to A at nucleotide position 19047; and

G to A at nucleotide position 20139;

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

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

The coronavirus may be IBV M41.

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

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

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

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

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

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

In a fifth aspect, the present invention provides a method for making the coronavirus according to the first aspect of the invention which comprises the following steps:

    • (i) transfecting a plasmid according to the fourth aspect of the invention into a host cell;
    • (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene;
    • (iii) allowing homologous recombination to occur between the replicase gene sequences in the plasmid and the corresponding sequences in the recombining virus genome to produce a modified replicase gene; and
    • (iv) selecting for recombining virus comprising the modified replicase gene.

 

The recombining virus may be a vaccinia virus.

The method may also include the step:

    • (v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from the recombining virus from step (iv).

 

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

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

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

Further aspects of the invention provide:

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

 

The disease may be infectious bronchitis (IB).

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

Vaccination may be by in ova vaccination.

The present invention also provides a method for producing a vaccine according to the seventh aspect of the invention, which comprises the step of infecting a cell according to the sixth aspect of the invention with a coronavirus according to the first aspect of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1—Growth kinetics of M41-R-6 and M41-R-12 compared to M41-CK (M41 EP4) on CK cells

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

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

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

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

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

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

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

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

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

FIG. 11—A) Snicking; B) Respiratory symptoms (wheezing and rales combined) and C) Ciliary activity of rIBV M41R-nsp 10,14 rep and rIBV M41R-nsp 10,16 rep compared to M41-CK (Bars show mock, M41R-nsp10,14rep; M41R-nsp10,16rep and M41-K from left to right of each timepoint).

DETAILED DESCRIPTION

The present invention provides a coronavirus comprising a variant replicase gene which, when expressed in the coronavirus, causes the virus to have reduced pathogenicity compared to a corresponding coronavirus which comprises the wild-type replicase gene.

Coronavirus

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

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

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

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

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

Coronaviruses are divided into four groups, as shown below:

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

 

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

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

IBV

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

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

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

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

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

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

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

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

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

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

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

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

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

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

 

IBV M41-CK Sequence 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. 11a-c).

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, 7 1Disturbed 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, (105 EID50 per dose).

The results (Table 😎 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 105 EID50 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; and
an 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; and
G 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; and
G 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.

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WO-2007/078203 July 2007 WO
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Patent History
Patent number: 10130701
Type: Grant
Filed: Jul 23, 2015
Date of Patent: Nov 20, 2018
Patent Publication Number: 20170216427
Assignee: THE PIRBRIGHT INSTITUTE (Woking, Pirbright)
Inventors: Erica Bickerton (Woking), Sarah Keep (Woking), Paul Britton (Woking)
Primary Examiner: Bao Q Li
Application Number: 15/328,179
Classifications
Current U.S. Class: Coronaviridae (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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Video: China chases the Coronavirus in the streets and turns stadiums into hospitals

February 04, 2020

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A clip from a clip showing anti-corona virus spray vehicles in Yichang, China

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Tweeters and Chinese media have published video clips and pictures of vehicles in Yichang, located in the Chinese province of Hubei, spraying an antiseptic against infection with the Corona virus.

Crottaz Finance@crofin67

Video shows #Chinese authorities spraying unknown chemicals in #Yichang city, #Hubei province. Not sure if good for your health.

And the Chinese CCTV channel published on its pages on the communication sites pictures of the spraying operations, accompanied by a comment that says: "A citywide operation, Yichang City has been treated with a disinfectant containing 8 tons of liquid containing 10 percent of sodium hypochlorite to curb the spread of the Corona virus."

"The city, which is about 400 kilometers from Wuhan (source of the virus), recorded more than 400 infections as of Tuesday," the channel added in its publication.

CCTV Asia Pacific@CCTVAsiaPacific

Replying to @CCTVAsiaPacific

當地時間2月4日21时,为了加强新型冠状病毒感染肺炎疫情防控工作,最大限度减少病毒滋生蔓延,湖北省宜昌市各相关部门和街道(乡镇)开始对城区道路、街面、桥梁、隧道等公共区域,以及老旧小区、农贸市场、城乡结合部等进行集中喷洒消毒。目前宜昌市有超过400例确诊病例。#武汉肺炎

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9:50 AM - Feb 4, 2020

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Chinese songwriter and journalist Kniking Chen, wrote on Twitter: "I've never seen my city before as I saw it tonight. The city has carried out a massive clean-up operation to contain the spread of the Corona virus."

Qingqing_Chen@qingqingparis

I have never seen my hometown like thisTonight, the city conducts full-scale disinfectant to contain #CoronavirusOutbreak, Yichang has recorded so far more than 400 confirmed cases as of Tuesday. The city is 400 kilometers away from #Wuhan. #2019_nCov

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9:41 AM - Feb 4, 2020

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Chen published pictures showing the Chinese authorities preparing runways and playgrounds to accommodate patients with the Corona Virus in Wuhan, despite the authorities' building of "Huanshan" and "Linshan" hospitals in Wuhan within six days.

Qingqing_Chen@qingqingparis

After Leishenshan and Huoshenshan specially designated hospital for treating #2019_nCov in #Wuhan, authorities are now turning several stadium & convention centers into new hospital to put ppl with less severe symptoms into quarantine. How do they look like?

The first hospital is "Huxhanshan" which is built on an area of 269 thousand square feet and can accommodate one thousand beds, and the second hospital, "Lichenshan" hospital with an area of 323 thousand square feet, can accommodate 1300 beds and is scheduled to open two days after the opening of the first.

The virus has killed more than 425 people and infected 20,000 in China, nearly all of them in the central province of Hubei, the epicenter of the virus, and it has spread to 25 countries since it appeared in December.

Chinese media said that the authorities began using small "Dron" drones to measure the temperatures of citizens living in high-rise buildings.

https://www.alhurra.com/a/الصين-ووهان-مكافحة-فيروس-الكورونا-عربات-رش-مستشفيات-داخل-المدرجات-ييتشانغ/532408.html

Edited by Butifldrm
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Here is an interesting link from Johns Hopkins that is tracking the virus cases, deaths, recoveries by country, city...stopping flights had to be done, but we don't know yet if it is in time, but we'll know in a couple of weeks

 

https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6

 

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China Pledged to Build a New Hospital in 10 Days. It’s Close

 

State news outlets reported that the 1,000-bed facility would accept patients from Monday even as construction workers raced to complete it

 

By Amy Qin

  • Published Feb. 3, 2020Updated Feb. 5, 2020
 
 
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Wuhan Institute Of Virology And Chinese Army Submit Patent For Gilead Anti-Ebola Drug To Fight Coronavirus

by Tyler Durden

Thu, 02/06/2020 - 17:35

 

Amid growing speculation that nCoV-2019, aka novel coronavirus, aka the virus behind the Wuhan Coronavirus epidemic, was developed in a lab (and not just any lab, but the BSL-4 rated "biological weapons" grade Wuhan Institute of Virology, located in... well, Wuhan) which was then released accidentally (or not) among the broader population - a thesis which China's official press is scrambling to refute even as more supportive evidence emerges by the day - we would like to point out that earlier this week, the Wuhan Institute of Virology, which as Nature in 2017 said studies the "world's most dangerous pathogens", has jointly with the Military Medicine Institute of the People’s Liberation Army Academy of Military Science, i.e. with China's Army, applied to patent the use of Gilead Sciences’ remdesivir to treat the current coronavirus outbreak.

remdesivir.jpg

The company has partnered with Chinese health authorities to run a Phase III clinical trial to assess remdesivir for treatment of the virus. The drug was originally developed to treat the Ebola virus, but wasn’t effective. According to preclinical assays the drug might be effective against the coronavirus, 2019-nCoV, as was published in the New England Journal of Medicine (NEJM). The drug was given to a U.S. patient for compassionate use on day seven of the disease and their condition improved on day eight.

The new clinical trial of an anti-Ebola drug to fight Coronavirus, will be conducted at Friendship Hospital in Beijing, China. The trial will enroll 270 patients with mild and moderate pneumonia caused by the virus.

“Gilead is working closely with global health authorities to respond to the novel coronavirus (2019-nCoV) outbreak through the appropriate experimental use of our investigational compound remdesivir. While there are no antiviral data for remdesivir that show activity against 2019-nCoV at this time, available data in other coronaviruses give us hope,” the company stated.

The Wuhan Institute submitted the patent application jointly with the Military Medicine Institute of the People’s Liberation Army Academy of Military Science. Researchers with both organizations noted in a paper published in Nature’s Cell Research this week that both remdesivir and chloroquine, used to treat malaria, may be effective in stalling the coronavirus.

 

 

 

That said, "even if the Wuhan Institute’s application gets authorized, the role is very limited because Gilead still owns the fundamental patent of the drug,” said Zhao Youbin, a Shanghai-based intellectual property attorney at Purplevine IP Service Co. “Any exploitation of the patent must seek approval from Gilead."

The Wuhan Institute indicated it filed the patent application on January 21 - days before China started officially releasing case information to the public, prompting questions how the Institute was so well prepared and knew just what the appropriate treatment against Coronavirus may be, but also noted it would temporarily drop the patent claims if it had the opportunity to collaborate with foreign biopharma companies to battle the epidemic.

Despite the recent breakout of market euphoria, where traders are now convinced that containing the pandemic is just a matter of time, the World Health Organization (WHO), however, is trying to downplay media reports of any drug breakthroughs against the outbreak, stating there are “no known” drugs against the virus. “There are no known effective therapeutics against this 2019-nCoV and WHO recommends enrollment into a randomized controlled trial to test efficacy and safety,” the organization stated today. “A master global clinical trial protocol for research and prioritization of therapeutics is ongoing at the WHO.”

 

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Gilead’s remdesivir is an experimental drug that isn’t licensed or approved anywhere in the world. It is being rushed into clinical trials in China. Gilead’s chief medical officer, Merdad Parsey, told Bloomberg that the drug could enter clinical trials in China as early as next week in patients with moderate and severe symptoms.

China can manufacture chloroquine and currently wants access to remdesivir. Bloomberg points out that the country’s decision to seek a patent “instead of invoking the heavy-handed ‘compulsory license’ option that lets nations override drug patents in national emergencies, underscores the delicate balancing act before China as it signals commitment toward intellectual property rights alongside curbing the virus outbreak.”

“The fact that they have applied for a patent means there’s growing awareness about this in the country,” said Wang Yanhu, a senior partner at Albright Law Offices in Beijing. “The government is compelled to avoid using the compulsory license because it has been making efforts to show China respects intellectual property rights and the abuse of compulsory licensing will draw international criticism.”

Gilead is presently shipping enough doses of the drug to China to treat 500 patients and is increasing its supply in case the clinical trials are effective.

https://www.zerohedge.com/health/wuhan-institute-virology-and-chinese-army-submit-patent-gilead-anti-ebola-drug-fight

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Virus Cases Explode On Quarantined Cruise Ship In Japan As China Claims Number Of Sick Is Shrinking

Profile picture for user Tyler Durden
Thu, 02/06/2020 - 19:36
 

Summary:

  • Total cases stand at 31,439 as death toll climbs to 639

  • China claims the growth in people under medical observation has peaked, and is now slowing.

  • Japan reports 41 additional cases, for a total of 61 aboard the quarantined cruise ship

  • Senior CCP official orders Wuhan to round up infected residents for mass quarantine camps

  • Beijing completes second coronavirus hospital in Wuhan

  • Wuhan hospital confirms that doctor who was one of first to warn about virus died on Thursday

  • Economists warn China faces difficult dilemma in deciding when workers should return to work

  • Taiwan halts visas for citizens of Macau and Hong Kong

  • Germany confirms 13th case

  • 2 suspected cases found in South Africa

  • Dubai reports 3rd death outside China

*  *  *

Update (1955ET): Just when you thought it was safe to buy the f**king record high melt-up, TBS News reports that Japanese authorities have found another 42 people on the Diamond Princess cruise ship anchored in Yokohama have tested positive for Coronavirus.

 
 
 

【速報】横浜に停泊中のクルーズ船内に新たに41人の新型コロナ感染者 厚労省 #指定感染症 #感染症 #新型コロナウイルス

View image on Twitter
 
 
 
 

Japan says 273 people on the cruise ship were tested and 61 were found positive, and the 41 new patients have been sent to hospitals in 5 separate prefectures.

*  *  *

Update (1925ET)China has just released its latest "official" data on the coronavirus, and just as we had suspected (given the precipitous slowdown from exponential growth yesterday), the acceleration has now 'officially' ended with 185,555 people under medical observation today, DOWN 309 from the 186,354 the prior day.

 

2020-02-06_16-23-49.jpg

Mission Accomplished? Bear in mind that the average increase for all prior days was 17,159.

Of course, the number of cases and deaths is still increasing with 637 dead in China and 31,161 cases.

 

2020-02-06%20%282%29.png

We wonder if the almost nationwide lockdown and policy shift to remaining in your home unless "severely" ill was the factor behind the slowdown in people receiving medical attention? Or whether it is a miracle that no 'expert' saw coming... apart from The WHO who all along claimed the "extreme measures" by China were "breathtaking."

*  *  *

Update (1615ET): A senior Chinese official has ordered Wuhan authorities to immediately round up all residents infected with the novel coronavirus and place them in isolation, quarantine camps, or designated hospitals, according to the New York Times.

wuhan%20quarantine.PNG Inside the exhibition center in Wuhan that now serves as a hospital.Credit...Chinatopix, via Associated Press

City investigators have also been ordered to go to each home and check the temperature of every resident, as well as conduct interviews with infected patients' close contacts.

"Set up a 24-hour duty system. During these wartime conditions, there must be no deserters, or they will be nailed to the pillar of historical shame forever," said Sun Chunlan, a vice premier in charge of leading the CCP's response to the outbreak.

The city’s authorities have raced to meet these instructions by setting up makeshift mass quarantine shelters this week. But concerns are growing about whether the centers, which will house thousands of people in large spaces, will be able to provide even basic care to patients and protect against the risk of further infection. -NYT

* * *

Update (1440ET): Dubai just reported that a woman from the Philippines has died in the UAE. In response, the UAE has suspended flights to China except to Beijing. The Manila Times reported the death, citing the Philippines' labor secretary.

The virus's global death toll has climbed to 568.

 

Screen%20Shot%202020-02-06%20at%202.40.5

The victim was identified as Amalia Collado Dapronoza, 58. Her death comes as 178 other patients remain under observation in the tiny Gulf kingdom. The Philippines is one of several countries planning to rescue its citizens from Wuhan, and a group of 45 filipinos from Wuhan are set to return to the Philippines on Feb. 9.

In Hong Kong, 3,600 people were preparing to spend a second night confined aboard the World Dream as authorities test everyone aboard after eight passengers caught the virus.  Health officials in the financial hub said they were also asking some 5,000 Hong Kongers who had traveled on the ship since mid-January to contact them.

We mentioned earlier that Beijing had finished building the second of two coronavirus hospitals built in Wuhan over the past week and a half. Well, here's a cool time-lapsed video of its construction:

 

China finished building the second of two coronavirus hospitals in just over a week. How they built them so quickly: https://www.wsj.com/articles/how-china-can-build-a-coronavirus-hospital-in-10-days-11580397751 

 
Embedded video
 
 
 
 

* * *

Update (1230ET): Beijing has finished construction on the  second coronavirus hospital in Wuhan - so hopefully more of the desperate patients stuck dying in their homes might soon have a bed at a public facility where their treatment can be overseen by doctors.

Chinese state media said the Leishenshan hospital, which will join the Huoshenshan hospital that opened Monday, will provide 1,600 beds and be staffed by 2,000 medical personnel. The project took roughly ten days.

 

Screen%20Shot%202020-02-06%20at%201.03.1

As we reminded readers earlier, the WHO appears to have gladly taken up the task of backing up Beijing's propaganda. But on Thursday, it confirmed a bit of bad news, saying that the doctor who was punished for his early warnings about the outbreak had succumbed to the virus. Now, Wuhan Central Hospital is denying that Li Wenliang, one of eight doctors who was punished by local police for his warnings, has died.

Instead, they said he was alive, but in critical condition, according to SCMP.

"In the fight against the pneumonia epidemic of the new coronavirus infection, our hospital's ophthalmologist Li Wenliang was unfortunately infected. He is currently in critical condition and we are trying our best to resuscitate him," it said in its official Weibo account.

Li, 34, an ophthalmologist at the Wuhan Central Hospital, was found to be infected with coronavirus on Saturday.

"We are very sorry to hear the loss of any frontline worker who is committed to care for patients...we should celebrate his life and mourn his death with his colleagues," said Michael Ryan, director of the World Health Organisation's health emergencies programme, said during a briefing on Thursday.

With Citadel's Ken Griffin becoming the latest to warn about supply chain disruptions related to the virus, Beijing is facing a difficult choice when it comes to deciding when workers should return to their desks or assembly lines.

In a separate SCMP story, economist Lu Zhengwei said allowing the workforce to return to their jobs was crucial both for supporting the economy and ultimately ending the epidemic.

"It’s obviously desirable for employers who are now paying rent, salaries and social welfare for their employees, for nothing in return," he said, adding that most small and medium enterprises in China could only last about a month in the current situation.

Meanwhile, a handful of new deaths have been confirmed by the SCMP:

 

Screen%20Shot%202020-02-06%20at%2012.57.

As the death toll climbs, doctors told the NYT that the number of deaths and cases are likely being undercounted.

Many doctors believe that the number of deaths and infections are undercounted because hospitals and laboratories are under severe strain to test for the virus. Local officials in Hubei, the center of the outbreak, have called on health care workers to speed up the process.

As we reported below, there have been many anecdotal reports about sick people being turned away by hospitals in Wuhan.

Advisory firm Oxford Economics has lowered its growth outlook for China to 5.4% in 2020, down from 6%, and Goldman analysts believe the outbreak will ultimately shave 2 percentage points off global GDP by the time it's all said and done.

Earlier, Taiwan halted visas for citizens of Hong Kong and Macau, saying on Thursday that it would suspend its online and landing visa services indefinitely for Hong Kong and Macau citizens, while non-citizens of Hong Kong and Macau with a history of travel to mainland China, Hong Kong or Macau would also be barred from visiting Taiwan. These measures will no doubt further infuriate Beijing, which has lashed out against "fearmongering" abroad.

"Beginning [midnight Thursday], we will suspend online and landing visa application services" until further notice, announced Chiu Chui-cheng, vice-chairman of Taiwan’s Mainland Affairs Council, which sets the island’s policies towards mainland China.

They're also temporarily banning cruise ships from docking - can't say we blame them.

More suspected cases of the virus have been discovered in Africa, though none have been confirmed yet. Still, African health officials have been scrambling to brace for outbreaks; they're hoping to suppress the virus before the outbreaks can overwhelm their fragile health systems, according to News24.

Two suspected coronavirus cases have been reported in KwaZulu-Natal although there was not yet any laboratory confirmation, the provincial health department said on Thursday.

Spokesperson Noluthando Nkosi said one case was being handled by a departmental facility, while the other was at a private facility in Durban.

"The department is monitoring the treatment and management of these patients quite closely."

Nkosi said the public would be notified should there be any new developments, urging people to "remain calm and avoid being misled by false statements which are peddled on social media platforms."

On Wednesday, a patient in Limpopo was cleared after being held in isolation at Ellisras Hospital in Lephalale.

In other news, Germany has confirmed its 13th coronavirus case, a case we imagine will also be found to be a human-to-human infection.

* * *

Update (1100ET): Is this the WHO's "Mission Accomplished?"

During a press conference in Switzerland Thursday evening, Dr. Tedros, the WHO's director-general, thanked the Gates Foundation (which committed $100 million) and Japan (which contributed $10 million) for answering the organization's call for more funding to help suppress the coronavirus outbreak that has now claimed more than 560 lives, mostly in China. He also claimed that the number of new and suspected cases is finally starting to slow, a sign that the outbreak could be subsiding, and that the China-led response has been a success.

Though researchers have already mapped the virus's DNA and are already working on a remedy, there is still a lot we don't know about the virus, Dr. Tedros said. We don't know where it came from (though there are theories) and we don't 100% understand how it spreads. There are also many tools we don't have, like a vaccine, that will hopefully soon be developed. In effect, "we're shadow-boxing with the virus," Dr. Tedros said. "We need to bring it into the light."

The organization continued to sidestep questions about China's censorship and its initial reluctance to share information about the outbreak with the international community.

Responding to a reporter's question, the WHO rep confirmed that the doctor who was one of the first to detect the virus before being ignored and eventually sickened has now died.

Dr. Li Wenliang, the Chinese whistleblower doctor who warned the public about the outbreak back in December, succumbed to the virus in Wuhan on Thursday, the WHO said.

* * *

Thousands of athletes around the world breathed a sigh of relief on Thursday when Japanese Prime Minister Shinzo Abe confirmed that the Summer Olympics in Tokyo won't be delayed. Then again, if the outbreak continues to worsen in Japan and the broader region, who is going to want to come if they don't feel safe?

As the second week of global pandemic panic comes to a close, China, increasingly frustrated that their ruse with the WHO didn't manage to calm the international community, again registered its "strong objections" to the growing number of travel bans directed at its citizens.

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Screen%20Shot%202020-02-06%20at%208.08.1

The warning followed a decision by Taiwan's health authority to ban all international cruise ships from docking at the island from Thursday as the number of suspected outbreaks aboard cruise ships grows.

The global death toll has ticked higher, reaching 566 overnight, while the total number of confirmed cases has broken above 28,000 to 28,384.

 

Screen%20Shot%202020-02-06%20at%208.05.0

 

2020-02-04_1_2.png

More than a dozen countries have imposed some kind of restriction on foreigners who have recently visited China. Within China, images of police clad in hazmat suites and touting infrared thermometers have become frighteningly common. Many airlines cancelled passenger routes to China, and some are extending those cancellations out to March or April.

"China is strongly concerned and dissatisfied," said a spokeswoman for China's Foreign Ministry. "We hope relevant countries will bear in mind overall relations and people’s interests and resume normal operation of flights to guarantee normal people-to-people exchange and cooperation."

"I must stress that certain countries’ ill-advised decisions to suspend flights to and from China are neither cool-headed nor rational," she added.

But while Beijing tries to spin the narrative to accuse other countries of racism, some brave journalists have shared the stories of families brave - or foolish - enough to speak out against the regime.

One resident of Wuhan who has been stuck in the city since the quarantine told the BBC that his uncle died in a quarantine because of supply shortages.

The image of life in Wuhan is every bit as bad as the most chilling conspiracies would have you believe.

"My uncle actually died in one of the quarantine points because there are no medical facilities for people with severe symptoms. I really hope my father can get some proper treatment but no-one is in contact with us or helping us at the moment."

"I got in touch with community workers several times, but the response I got was, 'there's no chance of us getting a bed in the hospital.'"

Beijing, which just announced a spate of new treatment-related projects in Wuhan and the surrounding area, seemingly can't get beds online fast enough. Because the government is literally condemns some elderly patients to die in their homes.

But for people like us, we can't even get a bed now, let alone get one in the new hospitals.

If we follow the government's guidelines, the only place we can go now is to those quarantine points. But if we went, what happened to my uncle would then happen to dad.

So we'd rather die at home.

Many are saying that if they knew authorities would lock down Wuhan last week, they would have left for the holiday earlier.

What I want to say is, if I knew they were going to lock down the city on 23 January, I would have definitely taken my whole family out, because there's no help here.

If we were somewhere else, there might be hope. I don't know whether people like us, who listened to the government and stayed in Wuhan, made the right decision or not.

In news from outside China, Indonesia is reportedly planning to build a quarantine center on an uninhabited island to isolate coronavirus victims, even though Indonesia has yet to record a single case of the virus, though 243 have been quarantined on the island of Natuna.

Across the globe, health officials are racing to develop treatments and testing methods for the virus. Wuhan, ground zero of the outbreak, opened an emergency test laboratory on Wednesday to begin human trials.

 

Screen%20Shot%202020-02-06%20at%208.07.0

Over in Hong Kong, a top public health official has declared a community outbreak, according to the SCMP.

A day after the city government revealed that it would impose a mandatory 14-day quarantine on anybody crossing into Hong Kong from China, the city government has provided some more details on how it will combat the crisis. Most of the new cases in the city are being caused by human-to-human transmission. Six people have been diagnosed with the coronavirus over the past few days, five of whom had not left the city recently. Of the 21 cases in total, eight are believed to have no travel history relevant to the coronavirus.

Circling back to the mainland, local authorities in the city of Tianjin announced on Thursday that it would ban the exit and entry of its villages and compounds, becoming the latest city to essentially quarantine its entire population. Over in Wuhan, authorities are now demanding that all residents report their temperatures at least once per day.

So, that's 60+ million people under quarantine in China. And though the pace of new cases in the country has slowed slightly, the virus is accelerating, especially in Asia.

https://www.zerohedge.com/geopolitical/wed-rather-die-home-chinese-citizens-rebel-against-mandatory-quarantine-lockdonw

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Chinese doctor censured for warning about coronavirus dies of the illness; death toll hits 636

 

https://www.usatoday.com/videos/news/2020/01/28/coronavirus-china-wuhan-disease-pandemic/4602147002/

 

 

 

 

A Chinese doctor who was reprimanded by security police for warning fellow doctors about the initial coronavirus outbreak has died of the illness, according to Wuhan Central Hospital.

Li Wenliang, 34, an ophthalmologist at Wuhan Central Hospital, was “unfortunately infected during the fight against the pneumonia epidemic of the new coronavirus infection,” the hospital said on its social media account. “We deeply regret and mourn this.”

He died early Friday despite an "all-effort rescue" after contracting the virus Jan. 30, according to The People's Daily, the newspaper of China's Communist Party.

The World Health Organization tweeted, “We are deeply saddened by the passing of Dr Li Wenliang. We all need to celebrate work that he did” on the virus.

199a6697-bbd2-4057-9267-85f24570d439-AFP

 
 

In a posting on social media, Li alerted fellow doctors Dec. 30 about the emergence of a SARS-like illness, warning them to wear protective clothing to avoid infection, the newspaper said. Wuhan is the epicenter of the coronavirus that has claimed 636 lives among more than 30,877 cases. The fatality rate is 2.1 percent; that compares to 9.6 percent for the SARS virus that created similar concern around the globe in 2002.

Four days later, Li was summoned by security police and forced to sign a 

letter that accused him of "making false comments" that had "severely disturbed the social order," according to the BBC. Police said he was one of eight people under investigation for "spreading rumors," the BBC reported.

Authorities later apologized to Li, who became a national hero as a whistleblower as the illness spread.

The chief epidemiologist at the Chinese Center for Disease Control and Prevention told the editor of the state-owned Global Times that China should praise the eight Wuhan whistleblowers. 

"They were wise before the outbreak," though any judgment needs to be backed by scientific evidence, Zeng Guang said.

 

We deeply mourn the death of #Wuhan doctor Li wenliang, who unfortunately got infected with novel #Coronavirus while battling with the epidemic. After all-effort rescue, Li passed away on 2:58 am, Feb. 7.

View image on Twitter
 
 
 
 

From his hospital bed, Li broke his police-imposed silence in late January, telling The New York Times in an interview by text message, “If the officials had disclosed information about the epidemic earlier, I think it would have been a lot better. There should be more openness and transparency.” 

Commentary on the social media account of the Supreme People's Court criticized the police for their actions, saying that although the initial reports by Li and others about a possible SARS outbreak were wrong, they were a useful alert that should have been allowed: "If the public listened to this 'rumor' at that time and adopted measures such as wearing a mask, strict disinfection and avoiding going to the wildlife market based on the panic about SARS, this may be a better way for us to prevent and control new pneumonia today." 

Contributing: The Associated Press

https://www.usatoday.com/story/news/world/2020/02/06/doctor-coronavirus-death-epidemic-wuhan-china-spread/4678344002/

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White House Asks Scientists To Investigate Whether 2019-nCoV Was Bio-Engineered

Profile picture for user Tyler Durden
Fri, 02/07/2020 - 17:26
 

A week ago, we published details that raised questions about the source of the Wuhan novel coronavirus, specifically questioning the official theory for the spread of the Coronavirus epidemic, namely because someone ate bat soup at a Wuhan seafood and animal market as a fabricated farce.

 

2020-02-07_10-07-43.jpg

The real reason behind the viral spread, we suggested, was that a weaponized version of the coronavirus (one which may have originally been obtained from Canada), was released by Wuhan's Institute of Virology (presumably accidentally ), China's only top, level-4 biohazard lab, which was studying "the world's most dangerous pathogens."

At the time we summarized the series of dots and asked "real reporters" to connect them:

 
 
  1. One of China's top virology and immunology experts was and still works at China's top-rated biohazard lab, the Wuhan Institute of Virology, which some have affectionately called the real Umbrella Corp.
  2. Since 2009, Peng has been the leading Chinese scientist researching the immune mechanism of bats carrying and transmitting lethal viruses in the world.
  3. His primary field of study is researching how and why bats can be infected with some of the most nightmarish viruses in the world including Ebola, SARS and Coronavirus, and not get sick.
  4. He was genetically engineering various immune pathways (such as the STING pathway in bats) to make the bats more or less susceptible to infection, in the process potentially creating a highly resistant mutant superbug.
  5. As part of his studies, Peng also researched mutant Coronavirus strains that overcame the natural immunity of some bats; these are "superbug" Coronavirus strains, which are resistant to any natural immune pathway, and now appear to be out in the wild.
  6. As of mid-November, his lab was actively hiring inexperienced post-docs to help conduct his research into super-Coronaviruses and bat infections.
  7. Peng's work on virology and bat immunology has received support from the National "You Qing" Fund, the pilot project of the Chinese Academy of Sciences, and the major project of the Ministry of Science and Technology.

Of course, that is all ancient history and Zero Hedge was permanently banned from Twitter for raising such a conspiracy theory about a publicly-searchable person working a publicly-searchable place.

 

china-virus-outbreak-700x420.jpg

But, bygones being bygones, we moved on... until today when no lesser entity than The White House began asking questions about the origin of the deadly coronavirus.

As ABC News reports, the director of the White House's Office of Science and Technology Policy (OSTP), in a letter to the National Academies of Sciences, Engineering, and Medicine, requested that scientific experts "rapidly" look into the origins of the virus in order to address both the current spread and "to inform future outbreak preparation and better understand animal/human and environmental transmission aspects of coronaviruses."

Specifically, ABC News' Chief Medical Correspondent Dr. Jennifer Ashton asked the director of the National Institute of Allergy and Infectious Disease about concerns that stem from misinformation online that the novel coronavirus could have been engineered or deliberately released.

"There's always that concern," Dr. Anthony Fauci said.

"And one of the things that people are doing right now is very carefully looking at sequences to see if there's even any possibility much less likelihood that that's going on. And you could ultimately determine that. So people are looking at it, but right now, the focus is on what are we going to do about what we have."

The White House has good reason to question the official story as expert virologist Dr. James Lyons-Weiler of the Institute for Pure and Applied Knowledge, showed and explained how the coronavirus' genetic sequence - which has been publicly released by China - contains a unique "middle fragment" encoding a SARS (severe acute respiratory syndrome) spike protein that appears, according to his genomic analysis, to have been inserted into the 2019-nCoV virus using "pShuttle" technology. This technique can only be done in a lab, as it has never occurred naturally in nature.

“This isn't a 'conspiracy theory', it is a scientific, medical theory,” says Del Bigtree, the Emmy-winning producer and founder of non-profit Informed Consent Action Network (ICAN).

“Its foundation is based on sound science, logic, and research, and the mainstream media needs to be asked whether it is going to pursue this link to SARS instead of trying to censor anyone who is trying to pursue truth via scientific fact.”

One additional, major reason we suspect this line of logical reasoning could be correct - as conspiratorial as it may seem - is that Chinese authorities have launched a full-scale crusade to disprove the fact that this deadly virus was man-made.

"Conspiracy theorists don't believe in science. I hope that the national professional department will investigate and give us an innocence," Shi Zhengli, a director at the Wuhan Institute of Virology - the only P4 level biosafety laboratory in China - responded to Caixin on February 4.

Faced with external doubts and accusations, on February 2nd, Shi Zhengli responded angrily in the WeChat circle of friends:

"The new coronavirus in 2019 is a punishment for uncivilized living habits of human beings by nature. It doesn't matter. I advise those who believe and spread rumours of bad media, believe in the so-called academic analysis that Indian scholars do not rely on, and close your mouth. "

Additionally, as The Washington Times reports, one ominous sign, said a U.S. official, is the that false rumors since the outbreak began several weeks ago have begun circulating on the Chinese Internet claiming the virus is part of a U.S. conspiracy to spread germ weapons.

That could indicate China is preparing propaganda outlets to counter future charges the new virus escaped from one of Wuhan’s civilian or defense research laboratories.

 

china_outbreak_06234_c0-209-5000-3124_s8

As a reminder, it was discovered that Shi Zhengli had participated in a study published in Natural Medicine on November 9, 2015, about a disease that can be caused by a SARS-like coronavirus (SHC014-CoV) found in Chinese horseshoe bats.

The researchers used the SARS reverse genetics system to generate and identify a chimeric virus. In simple terms, this chimeric virus consists of the surface protein of SHC014 and the backbone of the SARS virus. The chimeric virus can infect human respiratory cells, demonstrating that the surface protein of SHC014 has the necessary structure to bind to key receptors on cells and infect cells. Chimeras can cause disease in mice, but they are not lethal. Studies have shown that viruses currently circulating in bat populations could potentially trigger the potential risk of SARS-CoV (SARS virus) outbreaks.

We are sure this is all just a coincidence - Wuhan epicenter... Only P4 facility in China... A lab investigating bats infected with genetically engineered Coronavirus... Coronavirus infected Bats... Chimeric bio-engineered viruses.... and that is probably why The White House is now asking authorities to investigate the source of the disease.

Of course, when Zero Hedge suggested the same thing, highlighting key personnel in the Wuhan lab that were publicly acknowledged as responsible for that research, we were immediately banned by Twitter. We look forward to reading Buzzfeed's article decrying The White House for daring to ask questions about the origin of this deadly pandemic.

https://www.zerohedge.com/health/white-house-asks-scientists-investigate-whether-2019-ncov-was-bio-engineered

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Killer coronavirus is 'the worst enemy you can ever imagine' as viral outbreaks can pose a greater global threat than terrorism, World Health Organisation warns

  • WHO's director general warned coronavirus vaccine was still year and half away  
  • Dr Tedros Ghebreyesus said outbreak greater risk to humanity than terrorism
  • The epidemic has so far killed more than 1,100 people and infected over 45,000 

PUBLISHED: 04:06 EST, 12 February 2020 | UPDATED: 12:20 EST, 12 February 2020 

 

The deadly coronavirus outbreak is the 'worst enemy you can ever imagine' and more of a threat to humanity than terrorism, the World Health Organisation warns.

China hopes the killer virus, which has claimed more than 1,000 lives and struck down over 44,500 people, will be curbed by April.

But WHO's director general, Dr Tedros Ghebreyesus, said it could rumble on for more than a year and warned a vaccine could take at least 18 months to develop.

He added: 'To be honest, a virus is more powerful in creating political, social and economic upheaval than any terrorist attack. It's the worst enemy you can imagine.'

 
 
28.jpg?mode=stretch&connatiximg=true&scale=both&height=338&width=600

Yesterday, a top Hong Kong medical official predicted the coronavirus could infect more than 60 per cent of the global population if containment methods fail and the virus hits its spread potential. 

Professor Gabriel Leung, chair of public health medicine in the city, speculated on Tuesday that even if the coronavirus kills just 1 per cent of sufferers, it could still wipe out as many as 45million of the estimated 7.58 billion people on Earth.  

Health officials hope that faster identification of patients and containment methods will block the virus from reaching its deadly potential, but the outbreak has already sent shock waves through stock markets and supply chains around the world. 

 

See the entire tire article below 

 

https://www.dailymail.co.uk/health/article-7994665/Killer-coronavirus-poses-greater-global-threat-terrorism-World-Health-Organisation-warns.html

 

 

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Yes......watch your markets closely Pitcher.......my guess is if this really gets rolling we could see a 25-50% drop in the markets in a week......China has towns of 11 million totally closed up.......no production at all.......there will be a domino affect world wide to just that situation......at least IMO

CL

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The market was up big today on a report that the Coronavirus has peaked in China.  I have a hard time believing this, I’m just reporting what I saw today in the market. 

I had a hard time finding good patterns to Day Trade but did fairly well in Visa and Facebook. 

 

https://www.dailymail.co.uk/health/article-7996867/WHO-says-coronavirus-cases-stabilizing-China-outbreak-way.html

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During the Spanish Flu epidemic of 1918 the market dropped 40%

During the SARS epidemic of 2002-03 the Market dropped 30%.  

 

If if you want to see what could happen to the general market look at Oil and Energy stocks.  They are getting absolutely hammered. I read an article today that stated the Coronavirus has been a Black Swan for the Energy Business.  

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2 hours ago, Pitcher said:

During the Spanish Flu epidemic of 1918 the market dropped 40%

During the SARS epidemic of 2002-03 the Market dropped 30%.  

 

If if you want to see what could happen to the general market look at Oil and Energy stocks.  They are getting absolutely hammered. I read an article today that stated the Coronavirus has been a Black Swan for the Energy Business.  

Pitcher......as I understand it right now new daily reported cases are increasing 14-18% per day.....first it must slow....then decrease....

Thankfully most all like viruses to this.... usually subside during Spring and Summer....time will tell how serious this is....CL

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Those reported cases are the ones who come to a doctor.  Many don’t go.  I personally don’t believe a thing China reports. I have dealt with Chinese stocks for years and they are not very transparent.  It’s very hard to get the truth about their numbers whether it’s stocks or the Coronavirus.  

 

The thing that that is scary with this virus is the long incubation period. .  We’ll just have to see what happens.  I hope you are right and it subsides in the Spring .  There are several labs working on vaccines that should be available in 8 months to 18 months.  I know one thing for sure.  You could not get me on a cruise if you gave me a ticket.  

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11 hours ago, Pitcher said:

Those reported cases are the ones who come to a doctor.  Many don’t go.  I personally don’t believe a thing China reports. I have dealt with Chinese stocks for years and they are not very transparent.  It’s very hard to get the truth about their numbers whether it’s stocks or the Coronavirus.  

 

The thing that that is scary with this virus is the long incubation period. .  We’ll just have to see what happens.  I hope you are right and it subsides in the Spring .  There are several labs working on vaccines that should be available in 8 months to 18 months.  I know one thing for sure.  You could not get me on a cruise if you gave me a ticket.  

 

Pretty steep spike today.....my guess markets are going to take a pretty big hit in the next month or 2...     CL 

 

https://www.washingtonexaminer.com/news/coronavirus-infection-spikes-by-nearly-15-000-killing-an-additional-242

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Yesterday they report Coronavirus cases were peaking in China and the Stock Market goes up big.  Today the opposite is reported and the Stock Market is showing a pre Market way down.   

 

This is is why I am Day Trading this market.  How in the hell do you invest with this crazy volatility.  I don’t.  I’ll just wait for a better opportunity to go Long.  

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I read this morning the average 401K is over $300,000. That's a lot of billions someone is going to steal when the markets crash. I say that because the average person who holds a 401K will not have time to react and will take the hit. Today many Americans are feeling rich. What happens if they lose 40% or more? This could get ugly real fast.

 

B/A

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Market at all time highs.  One thing I never do is chase. Never never never.

 

i’ve always thought we would need a good Market correction so the rich could sack our retirement accounts before they push the RV button and have our global reset.   Don’t take my word as gospel.  I’m just rambling.  I’m sitting on my hands watching the market sputter.  I’m waiting patiently for a decent play.  Not seeing much so far.  

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It’s taken me years to develop the right scans, the right indicators, enter and exit disciplines, and chart settings for Day Trading. It’s also taken me years to develop the experience and patience to Day Trade and Swing Trade successfully.  

 

Today was a classic premarket drop on the methodology in counting infections of the Coronavirus in China.  The algorithms that read the news for the big funds do what they always do. They dump as soon as there is any bad news and the real traders sort thru the news to decide if they take it lower or the news is nothing to get to excited about.  Today they dumped em and bought em back on sale. They literally throw the baby out with the bath water on bad news.  

 

I got ahold of TWLO and had a nice trade.  I had several others but this was the best trade for me today.  A good Day Trader has to be patient and today I was and did pretty good.  Some days I’m spastic and break all my disciples.  It’s a tough gig but I like it.  

 

 

97D84401-F3FE-4E5B-BB6A-73D711E0CC5D.thumb.jpeg.c9cd16c84a6f81eb4b786b8560daaaed.jpeg

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      One of the big issues in the US is that very little testing is being done, and testing results aren’t being properly shared, which means we don’t know what’s actually happening. Scott Gottlieb, the previous FDA commissioner, explained that in Seattle there has been better testing, and we are seeing infection there: “The reason why we knew early about Seattle outbreak of covid-19 was because of sentinel surveillance work by independent scientists. Such surveillance never got totally underway in other cities. So other U.S. hot spots may not be fully detected yet.” According to The Atlantic, Vice President Mike Pence promised that “roughly 1.5 million tests” would be available this week, but less than 2,000 people have been tested throughout the US at this point. Drawing on work from The COVID Tracking Project, Robinson Meyer and Alexis Madrigal of The Atlantic, said:
      Part of the problem is that this has become a political issue. In particular, President Donald Trump has made it clear that he wants to see “the numbers” (that as, the number of people infected in the US) kept low. This is an example of where optimizing metrics interferes with getting good results in practice. (For more on this issue, see the Ethics of Data Science paper The Problem with Metrics is a Fundamental Problem for AI). Google’s Head of AI Jeff Dean, tweeted his concern about the problems of politicized disinformation:
      It doesn’t look like there is the political will to turn things around, when it comes to transparency. Health and Human Services Secretary Alex Azar, according to Wired, “started talking about the tests health care workers use to determine if someone is infected with the new coronavirus. The lack of those kits has meant a dangerous lack of epidemiological information about the spread and severity of the disease in the US, exacerbated by opacity on the part of the government. Azar tried to say that more tests were on the way, pending quality control.” But, they continued:
      Other countries are reacting much more quickly and significantly than the US. Many countries in SE Asia are showing great results, including Taiwan, where R0 is down to 0.3 now, and Singapore, which is being proposed as The Model for COVID-19 Response. It’s not just in Asia though; in France, for instance, any gathering of >1000 people is forbidden, and schools are now closed in three districts.
      In conclusion
      Covid-19 is a significant societal issue, and we can, and should, all work to decrease the spread of the disease. This means:
      Avoiding large groups and crowds Canceling events Working from home, if at all possible Washing hands when coming and going from home, and frequently when out Avoiding touching your face, especially when outside your home. Note: due to the urgency of getting this out, we haven’t been as careful as we normally like to be about citing and crediting the work we’re relying on. Please let us know if we’ve missed anything.
      Thanks to Sylvain Gugger and Alexis Gallagher for feedback and comments.
      Footnotes
      (Click ↩ on a footnote to go back to where you were.)
      Epidemiologists are people who study the spread of disease. It turns out that estimating things like mortality and R0 are actually pretty challenging, so there is a whole field that specializes in doing this well. Be wary of people who use simple ratios and statistics to tell you how covid-19 is behaving. Instead, look at modeling done by epidemiologists. ↩
      Well, not technically true. “R0” strictly speaking refers to the infection rate in the absence of response. But since that’s not really ever the thing that we care about, we’ll let ourselves be a bit sloppy on our definitions here. ↩
      Since that decision, we’ve worked hard to find a way to run a virtual course which we hope will be even better than the in-person version would have been. We’ve been able to open it up to anyone in the world, and will be running virtual study and project groups every day. ↩
      We’ve made many other smaller changes to our lifestyle too, including exercising at home instead of going to the gym, moving all our meetings to video-conference, and skipping night events that we’d been looking forward to. ↩
      This post is tagged: [ ai-in-society ] (click a tag for more posts in that category). Related Posts
      Disinformation: what it is, why it's pervasive, and proposed regulations 26 Feb 2020
      fastai—A Layered API for Deep Learning 13 Feb 2020
      Tech Ethics Crisis: The Big Picture, and How We Got Here 09 Feb 2020
       
    • By SocalDinar
      Focus
      Coronavirus underpins gold; $1,600 break would trigger chart buying
        Allen Sykora  Monday January 27, 2020 10:01   Kitco News
      Share this article:

      (Kitco News) - Gold futures have risen to their highest level in nearly three weeks, drawing a safe-haven bid amid worries about China's coronavirus spreading and hurting the global economy, as well as a weekend attack on the U.S. embassy in Iraq, traders and analysts said.

      Look for momentum-based chart buying to add further steam to the rally should these factors push gold above $1,600 an ounce again, one strategist said.
      As of 9:57 a.m. EST, Comex February gold was $6.90, or 0.43%, higher to $1,578.50 an ounce. The metal peaked at $1,588.40, its most muscular level since Jan. 8 when worries about a possible war between the U.S. and Iran prompted a safe-haven bid.

      "A big part is that is the coronavirus hitting stocks and possibly spreading," said Daniel Pavilonis, senior commodities broker with RJO Futures. "The market is trying to factor in what type of dent in global GDP [gross domestic product] from a slowing down in the world's second-largest economy and [the virus] spreading into other countries. It could be pretty big."

      All three of the major U.S. stock indices opened at least 1.8% lower on Monday.

      George Gero, managing director with RBC Wealth Management, commented he often describes gold prices as a barometer of geopolitics and the economy, rising when conditions are worsening, and vice-versa.

      "Now it is also a barometer of our well-being," he said, referring to the health scare boosting gold.

      While the virus is capturing the bulk of the headlines in the financial press early Monday, traders also listed to still other factors prompting a bid in gold. Phil Flynn, senior market analyst with Price Futures Group, cited the rocket attack on the U.S. embassy in Iraq.

      "There is a mixture of geopolitical risks, along with concerns about the fallout from the coronavirus and how that will affect capital flows going forward," Flynn said.

      Uncertainty about the U.S. impeachment process and Brexit may also be lending some support to the precious metal, Gero said.

      Flynn looks for gold to maintain a bid while the market continues to monitor the factors currently underpinning prices.

      "We should see the strength at least until midweek until we get more answers about the virus and the response [to the attacks] from the U.S.," Flynn said.

      Should gold top $1,600, however, momentum-based buying would likely be triggered, Pavilonis said.

      "At $1,600, it would become more technical," said Pavilonis. "Then I think you would see buyers come in on [an expectation] for more of a longer-term trend and buy, whether this virus fizzles out or not. We're getting close to the old highs."

      While gold is higher so far Monday, Commerzbank did characterize the virus as both a "blessing and a curse" for the yellow metal.

      Risk aversion has risen, the bank pointed out. Thus, gold is being bought as a "crisis currency" and safe haven, Commerzbank said. But the virus also may be curbing gold purchases in China, the world's largest gold-consuming nation, analysts continued.

      "While gold is in demand as a kind of insurance for financial investors – as can be seen primarily from the ongoing ETF [exchange-traded-fund] inflows – the coronavirus could depress physical gold demand in China," Commerzbank said. "Because public life there is grinding more and more to a standstill, some market observers expect less gold to be bought around the Chinese New Year's festival this year, which is normally a period of high demand."
    • By bigwave
      https://www.weaselzippers.us/442959-check-out-these-creepy-drones-spying-on-people-during-coronavirus-outbreak-in-china/
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