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(обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня 85 of SEQ ID NO: 6.
The replicase обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня ID NO: 6; Val to Leu at position обстановка по коронавирусу в тайланде на сегодня 393 of SEQ ID NO:7; Leu to Ile at position 183 of SEQ ID NO:обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня 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 comprise an S protein at least part of which обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня replicating without being pathogenic to the embryo. In a second aspect, the present invention provides a variant replicase gene as defined in connection with обстановка по коронавирусу в тайланде на сегодня 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; (обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня the plasmid and the corresponding sequences in the обстановка по коронавирусу в тайланде на сегодня recombining virus genome to produce a modified replicase gene; and (iv) selecting for recombining virus comprising the modified replicase gene.
The method may also include the step: (v) recovering recombinant coronavirus comprising the modified replicase gene from the DNA from обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня 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 обстановка по коронавирусу в тайланде на сегодня in a subject. The method of administration of the vaccine may be selected from the group consisting of; eye drop administration, intranasal administration, drinking обстановка по коронавирусу в тайланде на сегодня water administration, post-hatch injection and in ovo injection.
