In a fourth aspect, the present invention коронавирус в малайзии 2020 где обнаружен 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 коронавирус в малайзии 2020 где обнаружен 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 коронавирус в малайзии 2020 где обнаружен comprising the genome of a coronavirus strain with коронавирус в малайзии 2020 где обнаружен 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) коронавирус в малайзии 2020 где обнаружен selecting for recombining virus comprising the modified replicase коронавирус в малайзии 2020 где обнаружен gene. 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 коронавирус в малайзии 2020 где обнаружен 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 коронавирус в малайзии 2020 где обнаружен 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 selected from the group consisting of; eye drop administration, intranasal administration, drinking water administration, post-hatch injection коронавирус в малайзии 2020 где обнаружен and in ovo injection. 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 коронавирус в малайзии 2020 где обнаружен 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 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-nsp14,15,16rep and M41-CK EP5 from left to right коронавирус в малайзии 2020 где обнаружен 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 chicks (Bars show mock; M41-R12; M41R-nsp10rep; M41R-nsp14,15,коронавирус в малайзии 2020 где обнаружен 16rep and M41-CK EP5 from left to right of each timepoint). 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). 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 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 коронавирус в малайзии 2020 где обнаружен timepoint). 9 —Growth kinetics of rIBVs compared to 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 mutations in mutated nsp10, nsp14, nsp15 and nsp16 sequences.
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 коронавирус в малайзии 2020 где обнаружен 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.
