P1: OTA/XYZP2: ABC JWST211-c02JWST211-BridgenAugust 23, 201214:57Printer Name: Yet to ComeTrim: 244mm 168mm Part I Positive sense RNA viruses P1: OTA/XYZP2: ABC JWST211-c02JWST211-BridgenAugust 23, 201214:57Printer Name: Yet to ComeTrim: 244mm 168mm 2 Coronavirus reverse genetics Maria Armesto, Kirsten Bentley, Erica Bickerton, Sarah Keep and Paul Britton Avian Viral Diseases, Institute for Animal Health, Compton Laboratory, Newbury, Berkshire 2.1The Coronavirinae The Coronaviridae form part of the order Nidovirales, which comprises two sub- families, the Coronavirinae and Torovirinae. There are three genera of coron- aviruses, alpha-, beta- and gammacoronaviruses (Carstens, 2010), which were so named for their visual resemblance to the corona of the sun in negatively stained preparations (Figure 2.1) (Tyrrell et al., 1968). Representative members of each of the coronavirus genera are shown in Table 2.1. Possibly the most publicised coronavirus of recent years has been the human coronavirus SARS-CoV, which emerged in China in 2002 causing the severe acute respiratory syndrome epidemic (Drosten et al., 2003; Ksiazek et al., 2003; Marra et al., 2003; Rota et al., 2003). Coronaviruses have, however, been isolated from many vertebrates and cause sev- eral economically important diseases in livestock species, including pigs, cows and chickens, and domestic species, such as dogs and cats. Coronaviruses are enveloped viruses with a single-stranded positive-sense RNA genome of 2632kb, the largest genomes of all RNA viruses currently known, that replicate in the cytoplasm of infected cells. The genome associates with the nucle- oprotein (N), forming a helical nucleocapsid within the virus particles. Although common among negative-sense RNA viruses, coronaviruses are the only positive- sense RNA viruses to possess helical nucleocapsids, which are enclosed within lipid envelopes containing the spike (S) glycoprotein, membrane (M) protein and en- velope (E) protein (Figure 2.1). For general reviews, see (Siddell, 1995; Lai and Cavanagh, 1997; Enjuanes, 2005; Siddell et al., 2005; Enjuanes et al., 2006; Gor- balenya et al., 2006; Masters, 2006; Thiel, 2007; Britton and Cavanagh, 2008; Perl- man et al., 2008; Norkin, 2010). Reverse Genetics of RNA Viruses: Applications and Perspectives, First Edition. Edited by Anne Bridgen. 2013 John Wiley Cavanagh, 2005; Cavanagh and Gelb, 2008; Jones, 2010). Following an IBV infection, egg produc- tion and quality are impaired in layers, and weight gain in broilers is reduced (Cook and Mockett, 1995). Infected birds are predisposed to secondary bacterial infections such as colibacillosis and mortality in young chicks is not uncommon. Faecal ex- cretion of the virus is a consequence of replication in the intestinal tract; however, this does not normally result in clinical disease. Infectious bronchitis was fi rst described in the US in the 1930s (Schalk and Hawn, 1931; Beach and Schalm, 1936; Beaudette and Hudson, 1937) and is preva- lent in poultry farming across the world due to the intensive nature of poultry pro- duction, estimated to involve the global production of 55 billion chickens (50 billion broilers and 5 billion layers) on an annual basis. In a report, commissioned by De- fra in 2005 (Defra, 2005), IBV was indicated as a major cause of ill health among chickens and was implicated as being responsible for more economic loss in the UK P1: OTA/XYZP2: ABC JWST211-c02JWST211-BridgenAugust 23, 201214:57Printer Name: Yet to ComeTrim: 244mm 168mm 2.3 CORONAVIRUS GENOME ORGANISATION29 Table 2.1Coronavirus genera and species. GenusSpecies alphacoronavirusCanine coronavirus (CCoV) Feline coronavirus (FCoV) Feline infectious peritonitis virus (FIPV) Human coronavirus 229E (HCoV-229E) Porcine epidemic diarrhoea virus (PEDV) Porcine transmissible gastroenteritis virus (TGEV) betacoronavirusBovine coronavirus (BCoV) Human coronavirus HKU1 (HCoV-HKU1) Human coronavirus OC43 (HCoV-OC43) Human enteric coronavirus (HECoV) Murine hepatitis virus (MHV) Porcine haemagglutinating encephalomyelitis virus (HEV) Rat coronavirus (RtCoV) Severe acute respiratory syndrome coronavirus (SARS-CoV) gammacoronavirusIBV-like avianInfectious bronchitis virus (IBV) Turkey coronavirus (TCoV) Pheasant coronavirus (PhCoV) Non-IBV-like avianMunia coronavirus (MunCoV) Bulbul coronavirus (BuCoV) Thrush coronavirus (ThCoV) MammalianBeluga whale coronavirus SW1 (BeCoV) Asian leopard cat coronavirus OthersGoose coronavirus Pigeon coronavirus Duck coronavirus Note: The derivation of these species of gammacoronavirus has yet to be determined, according to the International Committee on Taxonomy of Viruses, Index of Viruses (Coronaviridae, 2008). poultry industry than any other disease (Bennett, 2003; Bennett and Jpelaar, 2005); IBV was estimated to cost the UK economy nearly19 million per year, mainly due to loss of egg production, with serious implications for animal welfare. The cost of control through vaccinati