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 коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 challenge associated with in ovo vaccination is that the virus must be capable of replicating in the presence коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 reduced pathogenicity for chickens, so that they can be коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 immune response is affected and the potential vaccine may not protect against the required serotype. In addition there коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 is a need for IBV vaccines which are not associated with these issues, in particular vaccines which may коронавирус последние новости на 2 февраля 2020 be used for in ovo vaccination. SUMMARY OF ASPECTS коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 коронавирус последние новости на 2 февраля 2020 following multiple passages in embryonated eggs because the position of each mutation is known and its effect on the virus, i.e. 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 коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 NO: 7; Leu to Ile at position 183 of коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 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 коронавирус последние новости на 2 февраля 2020 коронавирус последние новости на 2 февраля 2020 коронавирус последние новости на 2 февраля 2020 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 is from an IBV serotype other than M41. For example, the S1 subunit or the entire коронавирус последние новости на 2 февраля 2020 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.
