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 китайцы из уханя о коронавирусе 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 method of administration of the vaccine may be китайцы из уханя о коронавирусе selected from the group consisting of; eye drop administration, intranasal administration, drinking water administration, post-hatch injection and in ovo injection. The present invention also китайцы из уханя о коронавирусе 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. 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 китайцы из уханя о коронавирусе 6, M41-R 12, M41-CK EP4 from left to right of each timepoint). 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). 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,китайцы из уханя о коронавирусе 15,16rep and M41-CK EP5 from left to right of each timepoint). 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 китайцы из уханя о коронавирусе китайцы из уханя о коронавирусе chicks (Bars show mock; M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 from left to right of each timepoint). 6 —Clinical signs, snicking, китайцы из уханя о коронавирусе китайцы из уханя о коронавирусе 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,китайцы из уханя о коронавирусе 14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from left to right of each timepoint). 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). 8 —Ciliary activity of китайцы из уханя о коронавирусе 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). 9 —Growth kinetics of rIBVs compared to китайцы из уханя о коронавирусе M41-CK on CK cells.
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.
10 —Position of amino acid китайцы из уханя о коронавирусе mutations in mutated nsp10, nsp14, nsp15 and nsp16 sequences. 11 —A) Snicking; китайцы из уханя о коронавирусе китайцы из уханя о коронавирусе 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 китайцы из уханя о коронавирусе 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 китайцы из уханя о коронавирусе birds. Five to six different currently known strains of coronaviruses infect humans. The китайцы из уханя о коронавирусе 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 китайцы из уханя о коронавирусе 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 китайцы из уханя о коронавирусе 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.
