The present invention also provides a method for producing a vaccine коронавирус 18 02 20 коронавирус 18 02 20 коронавирус 18 02 20 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 коронавирус 18 02 20 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 коронавирус 18 02 20 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 of each timepoint). 5 —Ciliary activity of M41R-nsp10rep and M41R-nsp14,15,16rep коронавирус 18 02 20 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-nsp14,15,16rep 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 коронавирус 18 02 20 M41-CK (Bars show mock, M41R-nsp10,15rep1; M14R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,коронавирус 18 02 20 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,коронавирус 18 02 20 коронавирус 18 02 20 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 коронавирус 18 02 20 коронавирус 18 02 20 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 коронавирус 18 02 20 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, коронавирус 18 02 20 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 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 коронавирус 18 02 20 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 коронавирус 18 02 20 kilobases, which is the longest size for any known RNA virus. Coronaviruses primarily коронавирус 18 02 20 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 коронавирус 18 02 20 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 коронавирус 18 02 20 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 коронавирус 18 02 20 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 коронавирус 18 02 20 of minor clinical significance, but spontaneous mutation of this virus can result in коронавирус 18 02 20 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 коронавирус 18 02 20 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. Coronaviruses are divided into four groups, as shown below: Alpha Canine coronavirus (CCoV) Feline coronavirus (FeCoV) Human coronavirus 229E (коронавирус 18 02 20 HCoV-229E) Porcine epidemic diarrhoea virus (PEDV) Transmissible gastroenteritis virus (TGEV) Human Coronavirus NL63 (NL or New Haven) Beta Bovine coronavirus (BCoV) Canine respiratory coronavirus (CRCoV)коронавирус 18 02 20 —Common in SE Asia and Micronesia Human coronavirus OC43 (HCoV-OC43) Mouse hepatitis virus (MHV) Porcine haemagglutinating encephalomyelitis virus (HEV) Rat coronavirus (Roy). Rat Coronavirus is коронавирус 18 02 20 quite prevalent in Eastern Australia where, as of March/April 2008, it has been коронавирус 18 02 20 found among native and feral rodent colonies.
(No common name as of yet) (HCoV-HKU1) ?Severe acute respiratory syndrome coronavirus (SARS-CoV) Middle East respiratory syndrome coronavirus (MERS-CoV) Gamma Infectious bronchitis virus (IBV) Turkey coronavirus (Bluecomb disease virus) Pheasant coronavirus Guinea fowl coronavirus Delta Bulbul coronavirus (BuCoV) Thrush coronavirus (ThCoV) Munia coronavirus (MuCoV) Porcine coronavirus (PorCov) HKU15 The variant replicase gene of the coronavirus коронавирус 18 02 20 коронавирус 18 02 20 of the present invention may be derived from an alphacoronavirus such as TGEV; a betacoronavirus such as MHV; or a gammacoronavirus such as IBV. As used herein the term “derived from” means that the replicase gene comprises substantially the same nucleotide sequence as the wild-type replicase gene of the relevant coronavirus.
For example, the variant replicase gene of the present invention may have up to 80%, 85%, 90%, 95%, 98% or 99% identity with the wild type replicase sequence. The variant coronavirus replicase gene encodes a protein comprising a mutation in one or more of non-structural protein (nsp)-10, nsp-14, nsp-15 or nsp-16 when compared to the wild-type sequence of the non-structural protein. IBV Avian infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens which causes significant economic losses.
The disease is characterized by respiratory signs including gasping, coughing, sneezing, tracheal rales, and nasal discharge. In young chickens, severe respiratory distress may occur. In layers, respiratory distress, nephritis, decrease in egg production, and loss of internal egg quality and egg shell quality are common. In broilers, coughing and rattling are common clinical signs, rapidly spreading in all the birds of the premises. Mortality varies depending on age, virus strain, and secondary infections but may be up to 60% in non-vaccinated коронавирус 18 02 20 flocks. The first IBV serotype to be identified was Massachusetts, but in the United States several serotypes, including Arkansas and Delaware, are currently circulating, in коронавирус 18 02 20 addition to the originally identified Massachusetts type. The IBV strain Beaudette was derived following at least 150 passages in chick embryos. IBV Beaudette is no longer pathogenic for hatched chickens but rapidly kills embryos. H120 is a commercial коронавирус 18 02 20 live attenuated IBV Massachusetts serotype vaccine strain, attenuated by approximately 120 passages коронавирус 18 02 20 коронавирус 18 02 20 in embryonated chicken eggs.
H52 is another Massachusetts vaccine, and represents an earlier and slightly more pathogenic passage virus (passage 52) during the development of H120.
