Make no mistake: this disease is, in fact, a military biological weapon.
Two separate components of genetic sequencing from HIV-1, the virus which causes AIDS, were added to Bat-SARS-Like coronavirus in the laboratory, thereby allowing it to infect human lungs via the ACES2 receptors in our lungs, and to disrupt the human body ability to fight it off, by reducing human leukocytes.
The disease sequence from the original Bat coronavirus was uploaded to the National Center for Biotechnology Information at the US National Library of Medicine, in the year 2018 by China's Institute of Military Medicine, Nanjing Command. That Bat-SARS-Like coronavirus was issued Reference ID: AVP 70833.1 by the National Center for Biotechnology Information at the U.S. Library of Medicine.
The present outbreak of "novel Coronavirus" was uploaded to that same National Center for Biotechnology Information in January of this year by the Shanghai Public Heath Clinical Center and was issued Reference ID: QHD3418.1. Using the test facilities known as BLAST (Basic Local Alignment Search Tool) from the U.S. National Library of Medicine researchers have determined that the "virus envelope" of those two separate diseases, are 100% identical! We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019- nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag.
Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. There is no way in nature that the Bat coronavirus could fortuitously acquire the HIV genetic sequences, without causing a mutation of the Virus Envelope. The only way the virus envelope could obtain the HIV genetics and still remain 100% identical to the 2018 sample, is if the HIV genes were added in a laboratory.
So what the human race is now facing, is an accidentally-released military bio-weapon. The original Bat-SARS-Like Coronavirus was identified by China's People's Liberation Army, through the Institute of Military Medicine, Nanjiang Command in the year 2018. They uploaded the virus sequence and they were the sole entity in possession of the virus. Here we are, two years later, and the virus they had has been changed in a way that cannot occur in nature without mutating the virus envelope protein.
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Only in truth does charity shine forth, only in truth can charity be authentically lived. Truth is the light that gives meaning and value to charity. When Thai doctors applied AIDS and flu medicines together, they found improvements within 48 hours in patients infected with the Corona virus. This development can bring a solution to the Corona virus spreading from China.
ReplyDeleteThai doctors have declared the use of anti-flu and AIDS drugs a success in reducing symptoms of coronavirus patients after using them on a Chinese woman.
Two doctors from Rajavithi Hospital in Bangkok — Dr Kriangsak Atipornwanich, a physician, and Assoc Prof Dr Subsai Kongsangdao, a specialist — said at a briefing in Bangkok that improvements were seen in the 71-year-old woman 48 hours after administering the two groups of medications.
The woman was admitted first to Hua Hin Hospital and then transferred to Rajavithi Hospital on Jan 29.
After finding she had serious symptoms, the doctors decided to use oseltamivir, an anti-flu drug used to treat the Middle East respiratory syndrome coronavirus (MERS), as well as lopinavir and ritonavir, two AIDS drugs.
Dr Kriangsak acknowledged that the Chinese had already been using AIDS medications on novel coronavirus patients.
"We checked related information and found anti-flu drugs were effective on MERS so we combined both groups of medications.
“After poor lab tests for 10 days, the test finally turned positive after 48 hours of administering the medications. The treatment, as well as the recovery, is fast,” he said.
They noted they had reported the discovery to a medical journal and would use the drugs on additional patients. The two groups of drugs are effective on patients with serious symptoms, they said.
Coronavirus
ReplyDeleteJul 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.
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
ReplyDeleteThe 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.
ReplyDeleteThe 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.
DETAILED DESCRIPTION
ReplyDeleteThe 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.
RECOMMENDATIONS AND ADVICE FOR THE PUBLIC
ReplyDeleteDuring previous outbreaks due to other coronavirus (Middle-East Respiratory Syndrome (MERS) and Severe
Acute Respiratory Syndrome (SARS), human-to-human transmission occurred through droplets, contact and
fomites, suggesting that the transmission mode of the 2019-nCoV can be similar. The basic principles to reduce
the general risk of transmission of acute respiratory infections include the following:
• Avoiding close contact with people suffering from acute respiratory infections.
• Frequent hand-washing, especially after direct contact with ill people or their environment.
• Avoiding unprotected contact with farm or wild animals.
• People with symptoms of acute respiratory infection should practice cough etiquette (maintain distance, cover
coughs and sneezes with disposable tissues or clothing, and wash hands).
• Within health care facilities, enhance standard infection prevention and control practices in hospitals, especially
in emergency departments.
WHO does not recommend any specific health measures for travellers. In case of symptoms suggestive of respiratory
illness either during or after travel, travellers are encouraged to seek medical attention and share their travel history
with their health care provider.