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Avian Pathology
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The long view: 40 years of infectious bronchitis research


Jane K. A. Cook , M. Jackwood & R. C. Jones
a b a b c

138 Hartford Road Huntingdon, Cambridgeshire, PE29 1XQ, UK

Department of Population Health, College of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA, 30602, USA
c

School of Veterinary Science, University of Liverpool, Leahurst Campus, Neston, South Wirral, CH64 7TE, UK Accepted author version posted online: 02 Apr 2012.Version of record first published: 18 Jun 2012.

To cite this article: Jane K. A. Cook, M. Jackwood & R. C. Jones (2012): The long view: 40 years of infectious bronchitis research, Avian Pathology, 41:3, 239-250 To link to this article: http://dx.doi.org/10.1080/03079457.2012.680432

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Avian Pathology (June 2012) 41(3), 239 250

REVIEW

The long view: 40 years of infectious bronchitis research


Jane K. A. Cook1, M. Jackwood2* and R. C. Jones3
138 Hartford Road Huntingdon, Cambridgeshire PE29 1XQ, UK, 2Department of Population Health, College of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA 30602, USA, and 3School of Veterinary Science, University of Liverpool, Leahurst Campus, Neston, South Wirral CH64 7TE, UK
1

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The remit of this review is to provide the non-specialist reader of Avian Pathology with an overview of research carried out on infectious bronchitis over the 40 years since the journal was first published. In order to do this, we felt it necessary to summarize the knowledge acquired previously, since the since the disease was first identified in the 1930s. Infectious bronchitis virus is a significant pathogen in the domestic chicken, affecting the respiratory and renal systems as well as the female reproductive tract. The virus exists in the form of many, ever changing, serotypic or genotypic variants, some of which have global distribution whilst others are found only in more local areas. This review mentions the major discoveries concerning both the virus itself and the types of disease it causes and considers recent changes in its pathogenesis. It also discusses the impact of developments in the field of molecular biology and highlights possible areas for future work.

Introduction Infectious bronchitis (IB) is a highly contagious viral disease of the chicken. It is possibly the most economically important viral respiratory disease of chickens in regions where there is no highly pathogenic avian influenza virus (AIV) or velogenic Newcastle disease virus and is found everywhere that commercial chickens are kept. While initially a respiratory disease, the virus also affects the female reproductive tract, causing loss of production and poor egg quality. Some strains have a predilection for the kidney of young chickens, resulting in nephritis that can cause significant mortality. Control is attempted using liveattenuated and inactivated vaccines. IB was first described by Schalk and Hawn in the USA in 1931 as a respiratory disease of chicks and the viral cause established in 1936. Infectious bronchitis virus (IBV) is a coronavirus; an enveloped virus containing a single-stranded positive-sense RNA and projections called spikes on the surface of the virion that are involved in host cell attachment and induce neutralizing antibodies (Figure 1). The ability of IBV to genetically mutate and recombine results in antigenic shift and drift. This means that vaccine programmes are constantly being challenged. Thus, even though the disease was first reported more than 60 years ago, control is never complete even today. To celebrate the 40th anniversary of the journal Avian Pathology, this paper provides an overview of the history of research into IB, highlighting the major findings, assessing the current disease state and looking towards future research. Because this is not intended as a formal review we have not included full references since so many people have contributed to our knowledge of IB. More details can be found in the list of further reading included at the end of this brief review. The authors apologize to those colleagues whose work has not been mentioned specifically but who have nonetheless made a valuable contribution to our knowledge of this interesting and challenging virus.

Infectious bronchitis research prior to first publication of Avian Pathology The disease and pathogenesis general. Ciliated epithelial cells in the upper respiratory tract, epithelial cells in the female reproductive tract and tubular epithelial cells in the kidney are the main targets for IBV. For several years the virus has been tentatively linked with gut malfunction, and more recently it has been suggested as a cause of male infertility and venereal spread of virus from male to female. Respiratory tract. Clinical signs include depression, snicking, coughing, head-shaking and nasal and ocular discharge. Necropsy findings include tracheitis, inflammation of the lungs and thickened and frequently cloudy air sacs. Birds that die usually suffer asphyxiation due to caseous plugs in the bronchi resulting from secondary bacterial infection. In the 1940s and 1950s, histopathological studies performed after experimental infection by Hofstad in Iowa and by Jungherr and his colleagues at Connecticut demonstrated primary effects on the epithelial surfaces of the respiratory airways and frequently loss of ciliated epithelial cells accompanied by infiltration of subepithelial layers with lymphocytes and heterophils. In the lungs, the disease affects the bronchi in a similar way to the trachea; diffuse infiltration being the only other change. Tissue recovery is normally seen after 10 to 14 days.

*To whom correspondence should be addressed. Tel: '1 706 542547517. E-mail: mjackwoo@uga.edu Received 25 March 2012 ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/12/030239-12 # 2012 Houghton Trust Ltd http://dx.doi.org/10.1080/03079457.2012.680432

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the ovaries remained normal. Such birds have been termed false or silent layers. The proportion of a flock affected depended on the age at infection, in that at 1 day old the loss was 26%; at 18 days of age, 9.3%. A few years later Crinion and Hofstad in Iowa and Jones and Jordan in Liverpool, UK confirmed these findings. The American group found differences in effects between IBV types but could detect the development of oviduct abnormalities in the form of cysts as soon as 6 weeks after infection. Jones and Jordan showed that a pathogenic strain of the common Massachusetts (Mass) type could cause these effects in chicks with no maternal antibodies and, by immunostaining, attributed the bizarre oviduct development to virus replication in oviduct epithelium soon after infection (Figure 2). The kidney. From the 1950s, certain serotypes of IBV have been described as nephropathogenic, causing considerable swelling of that organ, with urates in the collecting tubules. This damage to the kidneys in young birds can result in significant mortality. The early work in this area was performed in the USA by Winterfield (Perdue University) and Hitchner (Cornell University), who described the Holte and Gray strains and used them to reproduce the disease in susceptible chickens. However, Cumming and his colleagues in Australia, working with the Australian T strain, did much of the pioneering work in this area. Kidney damage was the prominent clinical manifestation of Australian IBVs in the 1960s. Cumming showed that factors which predisposed chickens to the condition include chicken genetic line, dietary protein source, as well as chilling and other stress factors, and speculated that nephritis is a condition that perhaps all IBV strains are capable of inducing, given the required circumstances. In part, this was substantiated by the report of Jones in the next decade who described mortality due to nephritis and demonstrated virus in kidneys by immunostaining, in chicks accidentally chilled after infection with a not normally nephropathogenic Mass-type IBV.

Figure 1. Electron micrograph of infectious bronchitis virus negatively stained with phosphotungstic acid. Arrows indicate spikes on the surface of the virion. Bar 0100 nm

Female reproductive tract. The role of IBV in adversely affecting egg production and quality has long been known. It became apparent in the 1950s that two periods in the hens life are critical for the control of IBV: one at or after the point of lay and, less well appreciated, the second soon after hatching. The possibility that IBV could affect egg production and quality during lay was first studied by several American groups, including Delaplane and Stuart, and Fabricant. In the 1950s, Van Roekel and his colleagues in Massachusetts, in a 10year study of laying flocks, found that egg production drops varied widely, between 2 and 70% in affected flocks, and eggs laid subsequently had weak, cracked or misshapen shells with watery albumen. The likelihood of other complicating pathogens cannot be ruled out, but in a systematic experimental study a few years later, Sevoian and Levine in Georgia, USA induced a fall in egg production similar to that seen in field cases. They showed that the effects of the disease were more prolonged than simple disease-induced loss of food or water intake and that histological changes occurred in the oviduct in particular, which influenced the nature of eggs to be laid subsequently. Interestingly, IBV infection during the growing period appears to have little effect on the ability of the hen to produce eggs of normal quality. As early as 1957, Urban and Goodwin reported that infection at 11 to 12 weeks of age did not influence later production, while infection between 19 and 22 weeks of age inflicted serious egg loss, and Box in the UK found that infection at 18 weeks of age caused a fall of 35% but at 6 weeks of age the subsequent loss was 13%. All of these reports relate to different field or experimental conditions and virus strains, but the same picture emerges. At about this time, it became evident that with some IBV strains infection of female chicks soon after hatch could also be influential. Broadfoot and his colleagues studied the performance of 35 layer flocks with suboptimal production which all had respiratory disease within the first 3 to 5 days of life. IB was diagnosed by serum neutralization tests and egg production was reduced by up to 50%. Individual non-layers were found to have abnormal oviducts that were non-patent and cystic, so that eggs could not be laid externally, although

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Figure 2. Section of oviduct from a female chick 3 days after intranasal inoculation with Mass-type IBV. Immunouorescence stain shows virus in the epithelial cells. Bar 0 50 mm.

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Diagnosis of infectious bronchitis. IBV affects chickens of all ages, involving the respiratory, renal and reproductive systems, and because the clinical signs are not specific the need for differential diagnostic methods was quickly realized. These methods focus on either detecting or isolating the virus itself or detecting serum antibodies to it. Pioneering work in the USA by Cunningham (1970) at East Lansing and by Fabricant (1998) at Cornell University established that IBV grows well in embryonated eggs, and passage of field material via the allantoic route of 9-day-old embryos became the method of choice for virus isolation. The virus causes characteristic curling and stunting of the embryo, visible after about 7 days incubation and, after serial passage, embryo mortality may be observed with field strains. This method of diagnosis continued to be used for many years although, because of the need to rely on embryo stunting as an indication of IBV infection, and the frequent need for serial passage of field material before typical effects are seen, interpretation of the results was always subjective. Furthermore, the method is time consuming (7 days incubation being necessary before embryo lesions are observed) and expensive, requiring a significant number of embryos. However, passage in embryonating eggs is still in use to propagate the virus today. These issues made serological diagnosis seem a more attractive option, but again this was not simple in the case of IBV. In the early 1950s Fabricant developed standardized procedures for IB diagnosis and for interpretation of the findings using the serum neutralization test in embryonated eggs. In those days, the test was normally done using a single serum dilution tested against a series of dilutions of the virus, which added to the cumbersome nature of the test. In the early 1960s, Woernle in Germany pioneered the use of an agar gel precipitation test for IBV. Although used for some decades, probably because it is cheap and easy to perform, this test proved to be very insensitive and standardization of the test between different laboratories was never achieved. Its only merit seems to be that, because of the transient nature of precipitins, a positive result gives some indication of a recent infection. Whatever the diagnostic approach taken, there have always been difficulties to overcome because the virus affects so many tissues, making the choice of material for IBV isolation or detection critical. Since the primary target tissue for IBV is the upper respiratory tract, trachea or nasal tissue is the material of choice when investigating respiratory disease. However, the virus persists in the respiratory tract for a relatively short time after infection, and therefore kidney, or intestinal tract swabs or tissue, are the materials of choice when investigating kidney infection or poor egg laying performance. Interestingly, the reproductive tract has never been a productive tissue for isolation of IBV, perhaps because of the transient presence of virus there. Clearly IB diagnosis was an area in need of improvements and these were to come in the next decades. The immune response to IBV. The cross-neutralization test developed by Fabricant (see above) enabled the level of humoral antibodies post IBV infection to be detected and quantified. The test also allowed distinction between strains in terms of the degree of cross-neutralization using antisera against each strain. However, Raggis team at Davis in California was the first to demonstrate

a lack of correlation between titres of circulating antibodies and resistance to infection. Infectious bronchitis variants. The first realization that IBV was not homogeneous came with the observation by Jungherr in 1956 that an IBV isolated in Connecticut did not cross-protect chickens against challenge with the original Mass isolate. This led to the awareness of the existence of more than one IBV serotype or variant as they were to be called and had important implications for control. For many years this was thought to represent the first emergence of an IBV different from the Mass type. However, with the benefit of both hindsight and modern technology, we now know as a result of work by Naqis group at Cornell University that non-Mass-type IBVs have been present in the USA from the early 1940s and many more were subsequently reported. Vaccination against IBV. Attempts to control IBV were reported as early as the 1940s when Van Roekel showed it was possible to protect birds against the serious economic effects of IBV infection during the laying period by exposing them during rear to IBV that had been attenuated by passage in embryos. This was a very early example of the use of controlled exposure to protect against IBV and involved infecting a few birds with the virus and letting it spread naturally through the rest of the flock; a crude method but nonetheless effective. This approach also provided maternal antibodies to progeny, which were found to have some protective value initially. These observations gave impetus to the development of vaccination procedures. Early on, it was recognized that live-attenuated IB vaccines could be developed by reducing or attenuating the virulence of the virus by serial passage in embryonated eggs. Because IBV will only grow in cell culture once it has been adapted to do so by serial passages in the laboratory, the procedure of attenuating IBV by embryo passage, in use from the 1950s, is still employed today. In the early 1950s, the first IB vaccine was developed in the USA using the van Roekel M41 (Mass) strain. By the early 1960s IB had been diagnosed in The Netherlands, and this led to the development, by the Dutch veterinarian Bijlenga et al. (2004), of a vaccine based on an IBV isolated in that country and known as the H strain of the Mass serotype. The letter H stands for the name of the farmer (Huyben) from whose chickens the isolate was made and not, as is often thought, for the country, Holland. The resulting vaccines, known as H120 and H52, soon became widely used and the H120 vaccine is arguably still the most widely used IB vaccine worldwide today. The names give an indication of what is involved in attenuating an IB virus; H120 is a mild vaccine used from as young as 1 day of age and had received 120 embryo passages, whilst H52, for use only as a second vaccine, had received only 52 passages.

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Infectious bronchitis research 1970 to 1979 The first publications on IB in Avian Pathology were by Jordan and Nassar, looking at the survival of IBV in water, and by Hitchner, who examined the limitations of the virus neutralization (VN) test for classifying field isolates*both papers were published in Volume 2 (April

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1973). Subsequent publications during the 1970s addressed all aspects of the disease as well as diagnosis and control. The disease and pathogenesis. Renewed interest in the effects of IBV on the female reproductive tract was shown in this decade. Darbyshire and Cook, working at Houghton Poultry Research Station (HPRS) in the UK, found that co-infection with either Mycoplasma gallisepticum or adenovirus worsened the effects. A partial explanation for how IBV affects the oviduct was provided by Jones and Jordan, who demonstrated with immunostaining that after respiratory infection IBV replicates in all regions of the oviduct epithelium, and in the case of the pouch shell gland, to very high titre (Figure 3). The Liverpool work demonstrated that the effects on egg production of individuals varied greatly after identical respiratory application of the virus, with some birds out of lay for 3 to 4 days while others experienced a pause of more than 70 days. This group found substantial amounts of virus in the oviduct after IBV infections of hens, which posed the question of whether the virus is egg transmitted. In this decade, both McFerran at Stormont, Northern Ireland and Cook at HPRS found evidence of this when virus was isolated from chicks hatched from infected hens. However, in view of the significant reduction in the number of eggs laid by infected chickens and no reports of spontaneous outbreaks of IB in isolated flocks, this phenomenon, if it happens, seems likely to be of little importance. The intestine. For the first time, interest was shown in the possible role of the intestinal tract in IBV infections. Several papers in the 1970s by Alexander and his colleagues at the Central Veterinary Laboratory (CVL), Weybridge, UK and by Cook mentioned excretion of IBV strains in the faeces after cloacal swabbing, sometimes associated with apparent long-term persistence of virus in individuals or in the flock. The significance of enteric excretion has never been fully determined, except that it contaminates the environment, but the survival of IBV in faeces is poorly understood. Over the subsequent decades, various

reports described isolation of IBV from the intestines of experimentally infected chickens; and in the 1990s, Jones and colleagues confirmed this using immunostaining to show virus in villus tips at different regions in the gut, but with no apparent effect on gut function. Diagnosis of infectious bronchitis. With the identification of new IB variants came the need for improved diagnostic methods to identify them, but developments continued to be hampered by the need to grow IBV in embryonated eggs. Progress came in the 1970s after IBV had been adapted to grow in cell culture, and Hopkins in Georgia, USA was able to show that plaque-purified IBVs could be compared in that much simpler system. Hopkins work enabled both field isolates and sera collected from disease outbreaks to be studied in plaque reduction tests in cell culture. At about the same time, Johnson and Marquardt in Maryland developed the technique of performing VN tests in tracheal organ cultures using a constant amount of virus and varying dilutions of serum. This led to improved, quicker and cheaper methods for IB diagnosis and for differentiating IB variants. Following the observation that some IBV strains, when treated with the enzyme phospholipase C, were able to haemagglutinate chicken erythrocytes, Alexander and his colleagues developed a standardized procedure for performing the haemagglutination inhibition (HI) test for IB serology. Standardization was found to be very important because of the variable introduced by the need to enzyme-treat IBV before use. The test became widely used in IB diagnosis, although Bracewell and Brown at CVL demonstrated its limitations for differentiating between variants due to the high level of crossreactions found between them. The HI test continues to be used and limited differentiation of variants may be possible provided good controls are included and experienced technicians perform it. Immune response to IBV. The 1970s saw the publication of several papers beginning to investigate the role of antibodies in the immune response to IBV. The availability of the HI test enabled Gough and Alexander at CVL to confirm the lack of correlation between antibody levels and resistance to disease in the respiratory tract. However, humoral antibodies were shown to correlate well with absence of virus recovery from the kidney and genital tract (i.e. remote tissues). The importance of B cells in IBV infections was studied by Chubb in Australia, by depletion experiments using testosterone or cyclophosphamide. B-cell-depleted chickens showed increased clinical signs and more severe lesions, especially in the kidney. Infectious bronchitis variants. By the early 1970s, several different IB variants had been described in the USA, mainly using serology, and awareness of the problems that different IBVs could cause was beginning to be recognized. At that time almost all of the work in this area was being done by groups in America, including Johnson and Marquardt, Hitchner, Hofstad, and Winterfield, and important variants identified included Florida, Clark 333 and Arkansas. IB was reported for the first time in South America in the mid-1970s, and in both Brazil and Chile during this decade. Work done by

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Figure 3. Section of magnum of a mature hen 6 days after intranasal inoculation with M41 Mass-type IBV. Immunouorescence stain shows virus in the epithelial cells. Bar 0 25 mm.

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Lohr demonstrated that IB variants were certainly present in Malaysia in the 1970s. There was thus increasing awareness of different types of IB, and in the 1970s Cunningham, one of the pioneers of IB research, was the first person to attempt to group IB variants according to the immune response they induced. Vaccines. By the early 1970s, vaccination against IB was widely practiced and the live-attenuated vaccines H120 and H52 were almost universally used worldwide, except in the USA where Mass-type vaccines based on the M41 strain were developed. Importantly, Davelaar and Kouwenhoven at the Doorn Institute in The Netherlands showed that maternally derived antibodies appeared to have no adverse effect on the efficacy of live-attenuated IBV vaccines given at 1 day of age. However, the inability of Mass-type vaccines to protect against all of the new IB variants that by now were emerging gave impetus to moves to develop vaccines to some of these new variants. It will be clear from what has been said elsewhere in this review that the number of new IB variants being described was too large for a new vaccine to be developed against each one. Furthermore, it was soon realized that many of the variant viruses identified in commercial poultry disappeared so quickly that by the time a new vaccine had been produced and tested, the problem strain had gone away and been replaced by a new one. The skill was therefore in deciding which variant was of sufficient importance to warrant a specific vaccine. Vaccines developed in the USA at that time included Florida, Arkansas, and Connaught & Holland (both Mass types).

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commercial ELISA kits soon became available and continue to be widely used for IB diagnosis. These tests give a large amount of information very quickly and are invaluable for flock profiling and for monitoring responses to vaccination. However, they do have their limitations. The large amount of data rapidly generated means that careful interpretation of results is crucial and it is important to remember that ELISAs detect groupspecific rather than type-specific antibodies. Nonetheless, ELISAs continue to be the method of choice for serological monitoring for IBV. Another big advance in this decade came with the production of monoclonal antibodies (mAbs) for use in IB research, and these soon found many applications. For example, Koch and his colleagues at Lelystad in The Netherlands prepared a series of mAbs to IBV, which were specific for different structural proteins of the virus. On the basis of their reactivity to a panel of mAbs specific for the spike protein, clear differentiation between IBV field strains was possible, providing a much quicker method for this purpose than neutralization tests. However, a major limitation of the use of mAbs for the differentiation of IBVs is that a small mutation in the virus can lead to an amino acid change in the epitope with which a specific mAb reacts, leading to a misdiagnosis. Immune response to IBV. The work of MacDonald and colleagues in Edinburgh confirmed the protective effect of antibodies on remote tissues such as the oviduct, and this was echoed by Box who showed that IBV-specific antibodies were important in preventing drops in egg production due to IBV, by protecting the oviduct from viraemic virus. Vaccination studies have almost always focused on humoral immune responses in relation to protection. Nevertheless, Darbyshire at HPRS illustrated lack of correlation between antibodies and resistance, and discrepancies between in vitro strain differentiation by VN tests and in vivo cross-protection results. Furthermore, it was shown by Jones and Ambali that, following 1-day-old infection, re-excretion of virus at onset of lay can occur in the presence of high titres of circulating antibodies. All this suggested that while antibodies play a role in recovery from IB, other immunological mechanisms are involved. Immunity studies were further advanced during this decade, and possibly for the first time the importance of local and cell-mediated immunity in responses to IBV infections was realized. That the S1 (spike) glycoprotein of IBV induces VN and HI antibodies was established by Cavanagh et al. (1988) at HPRS and by Niesters at Lelystad, and this was considered the most probable inducer of protection at this time. The role of different classes of immunoglobulin (Ig) was also recognized in this decade. The major circulating immunoglobulin detected by HI was found to be IgG, and sensitive ELISAs were developed to measure it. Mockett and Darbyshire showed that anti-IBV IgG could be detected as soon as 4 days post infection, reaching a peak at 21 days and remaining at high titre in serum for many weeks, making this a useful measure of IBV infection and vaccine application. In contrast, antiIBV IgM was shown by Mockett to be present only transitorily, reaching a peak at about 8 days and then declining. This finding was later proven to be a useful

Infectious bronchitis research 1980 to 1989 Long-term persistence of IBV in the chicken. Although in the 1970s Alexanders and Cooks groups had reported on the shedding of IBV in faeces for many weeks after experimental infection, a true latency, such as occurs with infectious laryngotracheitis herpesvirus residing in the trigeminal ganglia, was never confirmed. However, in the 1980s work at Liverpool showed that after infection of 1-day-old female chicks, while virus could not be detected during the growing period, one strain of IBV was re-excreted sporadically at point of lay via both the respiratory tract and the gut. This occurred in the presence of high titres of circulating antibodies, but without clinical disease. Hormonal activity at sexual maturity was thought to be responsible for virus recrudescence but oestrogen and progesterone injections, designed to induce early sexual maturity, failed to bring forward virus excretion before 20 weeks of age. Diagnosis of infectious bronchitis. At the beginning of the 1980s the serum neutralization test was widely used for detecting and differentiating IB variants in serum, since this could not be done satisfactorily by the HI test. However, even when performed in cell or organ cultures, rather than embryonating eggs, it was still expensive and time consuming. A big step forward therefore came with the introduction of enzyme-linked immunosorbent assays (ELISAs), which were initially developed for IBV by Marquardt and co-workers and proved to be highly sensitive, allowing early detection of IBV infections and needing only small volumes of sera. A number of

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marker for recent infection, and ELISAs were developed to detect specific IBV IgM responses. Studies on local immunity centred on investigating the presence of IgA in lachrymal fluid, and the work of Davelaars group showed the importance of the Harderian gland. IgA is particularly important in responses to 1-day-old vaccination, and removal of the gland caused a decrease in protection. IgG could also be found in tears but was mainly serum derived. Mockett reported that maternal antibodies could be detected in tracheal washes but these were shown to be short lived. The first report of a possible role for cell-mediated immunity to IBV was by Timms and colleagues at CVL. They demonstrated antigen-specific proliferation of T lymphocytes in IBV-infected chickens. It was also shown using mouse mAbs that CD4 and CD8 antigens are found on T-helper and T-cytotoxic/suppressor T cells, respectively. In Australia, Chubb demonstrated the presence of cytotoxic lymphocytes in the spleen and peripheral blood. At this time the only cytokine studied with regard to IBV was interferon gamma. Otsukis group in Japan detected variable levels, mainly in respiratory organs, but the UK workers Holmes and Darbyshire could not detect it. Interestingly, there seemed to be little interest in determining what role, if any, interferon has in response to IBV infections, until very recently when Collisons group in Texas addressed the interferon gamma question and concluded that both natural and recombinant forms delay disease onset and decrease severity. Infectious bronchitis variants. Largely as a result of the work done by Kouwenhoven and Davelaar at Doorn, different IBVs suddenly became a big issue in Europe. A number of different variants (each given the prefix D) were described associated with disease problems in vaccinated flocks in The Netherlands and neighbouring countries. Shortly afterwards, Cook and Darbyshire developed the use of tracheal organ cultures as their preferred system, rather than embryonated eggs, for isolating and growing IBV and for performing crossneutralization tests. They subsequently isolated many different IB variants in the UK and other countries worldwide; many of which were not controlled by existing IB vaccines. This really was the decade of new IB variants. Over the next few years, similar as well as different variants were being isolated throughout Europe and this led to renewed interest in improving control measures to combat them. That this was a worldwide problem became clear in the 1980s with reports of IB variants from all continents and probably all countries with a developed poultry industry. In the USA, Hofstad, Gelb Jr (University of Delaware) and King (Southeast Poultry Research Laboratory, USDA) were examining the cross-immunity of numerous IBV isolates. One of the most significant discoveries was the isolation of the Arkansas-DPI virus from broilers on the Delmarva Peninsula; this virus became the parent strain for all of the Ark-DPI-type vaccines currently used in the USA. By the end of the 1980s, researchers were becoming aware that the incidence and distribution of IB variants was complex. Some viruses, such as Mass, occurred worldwide, the Arkansas virus appeared to be restricted to certain regions of the USA, whereas others such as D274 were found in Europe but not in the USA. The

occurrence of new IB variants and their incidence and distribution around the world is still unpredictable and confusing today. For further information, see the review by de Wit et al. (2011). Vaccines. Live-attenuated infectious bronchitis vaccines. Up to this time, most IB vaccines were based on the Mass type. Interestingly, during the 1980s it became clear in the USA that H120 vaccines from different sources provided different levels of immunity and had an unusual ability to cross-protect against some of the heterologous IBVs found there, including JMK and Florida. However, the emergence of an increasing number of variants led to calls for new IB vaccines to combat them. It has long been the case within the poultry industry that, if disease occurs in a flock, the blame is laid at the door of the vaccine itself, rather than consideration being given to whether or not that vaccine has been used correctly! However, it soon became clear that, whilst Mass-type vaccines could protect against some of the new types of IBV being found in both the USA and Europe, this protection was not complete and new vaccines were required to combat many of the new variants now being reported. During this decade, vaccines such as D274 and D1466 were developed in Europe and used there and elsewhere, in situations where the prevalence of a new variant has been established. Problems encountered in the USA at that time involved the Arkansas variant, which was established as a major problem in many of the poultry-producing areas. Inactivated infectious bronchitis vaccines. By this decade, it was realized that live-attenuated IB vaccines were not providing adequate protection throughout the life of layers and breeders, and furthermore could sometimes affect laying performance. This led to the development of inactivated vaccines for use in layers and breeders. Slow release of antigen and long lasting immunity throughout the laying period are achieved by intramuscular or subcutaneous injection of vaccines that incorporate an adjuvant together with IBV that has been inactivated by a method that destroys virus infectivity but maintains antigenic structure. Unfortunately, inactivated IB vaccines are not effective if used alone, so birds must still be given one or a series of vaccinations with live-attenuated IB vaccines during rear (live priming) prior to administering the inactivated vaccine. Other crucial problems had to be overcome in the development of inactivated vaccines, namely finding the optimal method of destroying the infectivity of the virus without losing its antigenicity and selecting the most suitable and effective adjuvant. Both of these issues caused continuing problems to researchers and vaccine manufacturers alike, being reflected in the high cost of these vaccines. Nevertheless, inactivated vaccines appear to be costeffective and continue to be used. Beginnings of research on molecular characterization of the virus. This decade saw the first interest in studying the structure of the virus itself. Structural characterization of the spike glycoprotein and nucleic acid sequence analysis of the viral RNA was being conducted by Binns et al. (1985) at HPRS and by Niesters and co-workers at Lelystad. The most significant finding was that neutralizing epitopes on the spike glycoprotein were encoded by hypervariable regions in the gene. These data laid the

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groundwork for the subsequent development of molecular-based diagnostic tests in the 1990s.

Infectious bronchitis research 1990 to 1999 Long-term persistence of IBV in the chicken. The site of persistence of virus has always been controversial, and Bhattacharjee and Dhinakar Raj working with Jones at Liverpool carried out work in this area. One series of experiments pointed to the kidneys as the probable site of persistence. After 1-day-old infection of chicks and long after clinical disease and virus excretion had ceased, virus could be reactivated by the T-cell immunosuppressor cyclosporin. In serial tissue examinations after treatment, virus first reappeared in the kidney, then in respiratory tissues, but was not detected in the caecal tonsil, considered by several workers to be the survival site. Dhinakar Raj argued that the kidney is an immunoprivileged site, where virus could survive away from the immune response. Diagnosis of infectious bronchitis. mAbs played a major role in the development of diagnostic techniques for IBV in this decade. Studies had begun on detection of different antibody classes in the response of the chicken to IBV and the importance of IgM in the early immune response to IBV infections was further recognised. Up to this time, different classes of antibody could be detected by affinity chromatography, but the availability of mAbs specific to IgM led to the development by da Silva Martins, then working at HPRS, of an IgM-specific ELISA for IBV. This technology was developed further by Naqi and by de Wit and his colleagues at Deventer, who devised an IgM-antibody capture ELISA specific for IBV. A different application of mAbs in IB diagnosis is illustrated by the work of Ignjatovic in Melbourne, Australia, who used mAbs directed against the three main structural proteins of IBV to detect and differentiate the IB variants found in Australia. Perhaps the most significant discovery of this decade was that molecular characterization of the spike gene correlated with serotype of the virus, which led to the development of molecular techniques to type IBV. In 1991 Lin and co-workers at the Nippon Institute in Tokyo, described the use of reverse transcriptase polymerase chain reaction (RT-PCR) to amplify a 400-basepair region of the spike, followed by restriction enzyme fragment length polymorphism (RFLP) as a typing method for IBV. Shortly afterwards, Jackwood et al. (2005) at the University of Georgia in Athens, USA described a similar procedure and showed that it could be routinely applied to field samples for rapid typing of IBV isolates. That assay employed primers that amplify the S1 gene of any IBV, followed by the use of three restriction enzymes to produce a unique type-specific pattern of DNA fragments on an agarose gel. The technique could be used for all IBVs. In addition, Jackwoods group discovered that phenol-inactivated viruses could also be typed. This very important finding allows IBVs from all over the world to be transported for identification. A different approach was described by Keeler et al. (1998), at the University of Delaware, where specific primers were designed and used to identify and differentiate six different serotypes of IBV. As nucleic acid sequencing became easier and less expensive, RFLP

analysis gave way to sequence analysis of the RT-PCRamplified spike genes. This was a vast improvement over RFLP analysis because sequences of the spike gene can be aligned and used to compare the relatedness between known strains and new variants (Figure 4). In addition, an explosion of sequence data for IBV spike genes from all over the world was being submitted to GenBank, allowing anyone with access to the Internet to compare the IBV types detected worldwide. Immune response to IBV. Further studies showing the importance of humoral antibodies in IBV infection by Cooks group using surgical bursectomy rendered an IBV-resistant chicken line (line C) more sensitive to infection, with an increased severity and duration of clinical disease. However, mortality rates did not increase, although humoral antibodies seemed to protect the tracheal epithelium following secondary challenge. Further work on local antibodies in the trachea, showing better correlation between lachrymal antibodies and protection, led Toros group at Auburn, Alabama to recommend that lachrymal antibodies could be used for antibody profiling of flocks. The same group later found that a light layer breed had a higher serum IgG and lachrymal IgA response than a heavy brown layer breed. Cooks groups meanwhile confirmed that chicken lines resistant to IBV had higher levels of IgA in tears than sensitive lines, although antibody titres in tracheal washes were similar. Local immunity in the oviduct was confirmed by Dhinakar Raj & Jones (1997), who detected virus-specific IgG and IgA in oviduct washes of IB-infected hens. In addition to local production, antibodies later transuded into the oviduct from the serum, but their value in oviduct protection remains to be determined. Using in vitro challenge of oviduct organ cultures from vaccinated chickens, the same group found in young chicks that local antibodies in the oviduct were less protective than those in the trachea. Regarding the intestinal tract, replication of IBV in the gut was earlier established by Lucio and Fabricant and by Ambali and Jones at Liverpool. Dhinakar Raj & Jones (1997) further demonstrated local antibody production in the duodenum and caecal tonsils of hens infected with an enterotropic IBV strain. Previously, Lutticken and others in the Netherlands failed to detect antibodies in gut washings after H120 and H52 vaccine application, but the differences may have been due to the use of different virus strains. The role of these antibodies in limiting gut replication needs further elucidation. Cell-mediated immunity to IBV was further explored in this decade. Janse and co-workers at Lelystad contended that immunity to IBV in the trachea is mediated by T cells. CD4 and CD8 cells were shown in tracheal sections by this group and by Dhinakar Raj & Jones (1997). However, while the Dutch group found an increase in CD4 cells, the UK workers found higher levels of CD8 cells. Again, these differences may have been related to virus strain. The Liverpool group also showed that after T-cell immunosuppression with cyclosporin, virus titres in kidneys were higher than in untreated birds, suggesting that T cells may also protect the kidney. Infectious bronchitis variants. With the development of molecular-based diagnostic typing tests, identification of

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J. K. A. Cook et al. Figure 4. Phylogenetic tree based on the amino acid sequences of selected strains of IBV. The neighbour-joining and Nei Gojobori methods were used to compute the relatedness. The amino acid sequences were aligned with ClustalW for IBV types: California 0CAV/CAV56b/91; Australia (subgroup 1) 0N1/62 S1; Georgia08 0GA08/HSp16/08 S1; Arkansas 0Ark/ArkDPI/81; D274 (European group B) 0B/D274/ 84; D3869 (European group E) 0E/D3869/84; 4/91 0793B/4-91/91; Georgia 07 0GA07/GA07/07 S1; QX 0QX/QXIBV/99; Massachusetts 0Mass/H52/S1, Mass/H120/S1,Mass/Mass41/41 S1; Connecticut 0Conn/Conn46/51 S1 vaccine; Florida 0FL/FL18288/71; Delaware 0DE/DE072/92 S1 vaccine; Georgia 98 0GA98/0470/98 S1; D1466 0Dutch/D1466/81.

a new IB variant became possible in days rather than weeks. Consequently, new genetic types were rapidly being identified worldwide, including in the USA, Central and South America, Europe, Australia and, particularly, in Japan, China, Taiwan and Korea. Some of the viruses from the Far East were shown to be closely related to ones already reported from Europe and the USA, but others appeared to be unique to particular countries and even to specific regions of that country. It was soon apparent that the situation was extremely complex and difficult to interpret. Were new variants suddenly emerging with much greater frequency, or was it simply that we were able to type many more viruses in a short period of time? We do not know for sure, but what we do know is that many of these variants, whilst readily detectable, do not seem to be of major significance in terms of the disease they caused, nor do they appear to persist for very long. Over the past 40 years, many IB variants have come and gone, but just a few have haunted the poultry industry for a long time. The Arkansas-type virus continues to be a problem in the USA and a cousin of that virus, the California variant first identified in 1991, still circulates in chickens there. In addition, the Delaware virus (DE072) identified in 1992 is still found in commercial poultry in the USA. Interestingly, the Arkansas, California and Delaware viruses have not been reported in countries other than the USA. However, this is not the case with the variant known variously as 793B; 4/91; CR88 (referred to here as 4/91). This variant, first reported in the UK and France at the beginning of this decade, is now found in many parts of the world, except, interestingly, the USA. The pathology caused by 4/91 was not typical of other IBV infections since it was originally associated with mortality in breeders, scouring in broilers and possibly muscle myopathy under field conditions. This variant continues to be a problem in many parts of the world, but it does not appear to have changed greatly in its structure or in its antigenicity, such that the vaccines which were originally developed to combat it still provide adequate control today. In Australia, several unique IBV types were identified which were only found in that country and were different from the nephropathogenic Australia T strain identified in the 1960s. These include the Vic S, N1/62, N9/74, N2/ 75 and V5/90 strains in Australian Group I and N1/88, Q3/88, and V18/91 in Group II. Finally, one notable IB variant identified in China was the QX variant. Unknown at the time, this virus eventually became perhaps the most significant IBV type apart from Mass in the years to come (see below). Vaccines. By the 1990s, the vast increase in the number of IB variants being identified led to major concerns about how best to protect against them. Developing a new vaccine against the multitude of new variants identified was clearly not possible. In addition, it takes several years to develop and licence a new vaccine, which in hindsight is probably a blessing since many new variants tend to be transient problems, coming and going in only a few months or years. During the 1990s Cook, building on the findings of Lohr, who showed that it was possible to group IBVs into protectotypes, demonstrated that much broader protection could be obtained than that suggested by serotyping or genotyp-

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ing. Based on analysis of ciliostasis in the trachea, Cooks work showed that, using a vaccination programme including a vaccine of the Mass serotype and one developed against the 4/91 variant, it was possible to protect against many, if not all, of the IB variants causing problems in many parts of the world. It is interesting to speculate whether this finding holds true for other different combinations of IB vaccines, or whether this is a property unique to these two particular strains. Certainly they are very different from each other genetically and are also both known to be strong immunogens. The precise mechanisms of this phenomenon have not been elucidated and this is an area that merits further research.

and silent layers in flocks in production, apparently resulting from infection at a very young age. This results in cystic lesions and, although the ovaries remain normal, eggs are not laid due to the severe and permanent damage to the oviduct. These effects have been reproduced experimentally by Benyeda and his colleagues in Hungary and by de Wit in The Netherlands. The rarity of occurrence of this form of IB suggests that not all IBVs have the same predilection for the oviduct, or perhaps that heterologous maternal antibodies cannot always neutralize viral infection in the first few days of life. Male reproductive tract. Although Cook in the 1970s detected IBV in the semen of infected cockerels, a finding that suggested venereal transmission could occur, this has been largely ignored until recently, when Bolz and co-workers in Illinois and Villarreal and her colleagues in Brazil detected IBV in the testes of cockerels in flocks where male infertility was suspected. Toro and co-workers recently confirmed the earlier finding of Cook and demonstrated IBV in the testis by immunostaining. Whether all IBVs have a tropism for the cells in the testis that produce spermatozoa remains to be determined, as does the importance of this finding. Long-term persistence of IBV in the chicken. Early in this decade Naqis group showed that IBV could persist and be shed in the trachea and cloaca of chickens for as long as 77 days. More recently, Jackwoods group has provided evidence in favour of the caecal tonsil being the site of IBV persistence, by detecting virus in this tissue using immunostaining several weeks after infection. Sufficient inconsistencies exist in the literature to indicate that perhaps the site of IBV persistence in the bird depends on the infecting virus strain and the age of the bird at infection. In small experimental groups it is usual to find that infectious virus is no longer detected a few weeks after infection. However, in commercial flocks of many thousands of birds, recycling of virus*a socalled rolling reaction*is likely to occur. Raj & Jones (1997) considered that long-term persistence might allow virus mutation, but work by Naqis group suggests that, for the Mass strain at least, it does not result in changes in the nucleotide sequence of the S1 gene or in tissue tropism. The mechanisms and frequency of long-term persistence in the epidemiology of IBV are unknown. However, it may have significance in that a clinically normal flock re-excreting virus could act as an unexpected source of infection. Diagnosis of infectious bronchitis. By this time, molecular biological techniques were becoming widely used in diagnosis. This technology enabled large-scale epidemiological surveys to be conducted elucidating the global distribution of IBV variants. The finding that dried swabs and, a few years later, FTA cards impregnated with material that inactivates viruses but still permits the detection of viral RNA by RT-PCR, provided a means to examine an enormous number of samples from all over the world. Thus, large amounts of spike gene sequence data quickly became available in GenBank. These data proved invaluable for comparing viruses, but it is critical to remember that in a diagnostic setting the technique detects viral RNA and gives no indication of

Infectious bronchitis research 2000 to 2011 Molecular biology. In 2002 the outbreak of severe acute respiratory syndrome (SARS) caused by a coronavirus in humans was significant for IBV research because a large number of laboratories and considerable resources were immediately targeted to research on coronaviruses. Many molecular characteristics of the virus were studied, including transcription and replication, protein synthesis and structure, virion assembly, mutation rates, recombination and viral genetic diversity, infectivity and epidemiology. The disease and pathogenesis. Tissue tropism. AbdelMoneim and co-workers in Egypt showed by immunohistochemical staining that, following inoculation of embryos with the M41 strain, IBV could be detected in the nasal epithelium, trachea, lung, spleen, myocardial vasculature, liver, gastrointestinal tract, kidney, skin, sclera of the eye, spinal cord, as well as in brain neurons. These results were consistent with virus isolation and denote the wide tissue tropism of M41 in the chicken embryo, perhaps reflecting the relatively undifferentiated state of the embryonic cells, rather than confirming an almost pan-tropism for chicken tissues, which has not been demonstrated in the hatched chick. The importance of Australian IBV research in this decade is demonstrated by the work of Ignatovic and her colleagues, who interestingly showed in a 20-year study of Australian IBVs that, over time, the kidney tropism, historically a consistent characteristic of IBVs in Australia, had changed to one for the respiratory tract and that this had been due to a small sequence change in the S1 gene. Effect of IBV on the female reproductive tract. In the last decade, notable work by Chousalkar and colleagues in Australia has shown detailed differences between strains of IBV in pathogenicity for the oviduct, including the finding, using real-time RT-PCR, that some strains can persist in that tissue for longer than was first thought, with a peak between 10 and 14 days post infection. The phenomenon of silent layers, first described in the 1970s, was largely forgotten since it had been seen rarely over the last few decades, but it emerged again in this decade with a vengeance following the appearance of the QX genotype of IBV in Europe at the beginning of the last decade. Although this variant was first reported in China associated with proventriculitis, when it arrived in Europe it was found to cause nephritis in young birds

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whether or not live virus is present in the bird at the time of sampling. This is an important epidemiological point when trying to correlate the results of RT-PCR surveys with the disease situation in the field. Furthermore, the techniques do not provide live virus, which may be needed for vaccination and challenge studies. Immune response to IBV. This decade saw little major work on the more basic aspects of immunity to IBV. However, there are a few studies that warrant mention. Secondary bacterial infection following an initial IBV infection has always been a major concern, and Vervelde and colleagues in Utrecht, The Netherlands examined colibacillosis and asserted that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function. The importance of IBV-specific maternal antibodies was illustrated by De Herdt and his colleagues in Ghent, Belgium, who showed that economic losses associated with IB in broilers were found predominantly where maternal antibodies were low and erratic. The interaction of IBV with other important pathogens in the chicken has always been a concern, and the immunosuppressive effects of chicken anaemia virus and infectious bursal disease viruses on local antibodies to IBV infection were clearly shown by Toros group. Both viruses caused a reduction in IBV-specific IgA in the respiratory tract, thereby confirming the major role that immunosuppressive agents may play in the chickens response to infection with IBV. Cell-mediated immunity. This was further studied by Collisson and her colleagues in this decade. They showed that memory T cells can be detected in blood for at least 10 weeks after IBV infection, while virus-specific CD8 memory cells can protect syngeneic chicks from acute IBV infection. Further work on in vitro stimulation with IBV antigen showed that B cells can be activated to secrete antibody by 3 weeks post infection. It was asserted that detection of such activated cells could be useful in early detection of infection. Wangs group in South Dakota followed the gene transcription profile of tracheal epithelial cells three days after infection of chickens with an attenuated IBV Mass strain. They confirmed that a diversity of innate immunity and helper T-cell-type-1-biased adaptive immunity are activated in the hosts early defence against IBV invasion and are responsible for the rapid clearance of virus from the local infection. Innate immunity and other factors. In the past decade, several novel, sophisticated techniques have been applied to research into IBV and have increased our knowledge of this virus and of immune responses to it. They have included aspects of innate immunity, immune modulators and genetic resistance. Some examples of these publications are mentioned here: Niu and co-workers in Dalian, China reported that the innate response to IBV could be stimulated by injection of baculovirus, which enhanced inflammatory cytokine mRNA expression in neonatal chicks. Similar to vaccination developments in other species, the use of immune-modulating agents has been reported. Babiuk and colleagues in Saskatchewan showed that CpG oligodeoxynucleotide is able to limit

IBV propagation in embryonic tissues, and they suggest that it might play a part in future vaccine design. With regard to genetics of the chicken, Etches and colleagues in East Lancing reported that the genes tightly linked to the B haplotype are those relevant to IBV resistance. Finally, Wangs group, in a further report on the complexity of immunity to IBV, contended that a number of innate immune factors of mucosal immunity are activated after IB immunization, including Toll-like receptors, type 1 interferons, interleukin 1 beta, complement, T-cell signalling molecules, surface markers and effector molecules. Infectious bronchitis variants. During this decade, many new variants continued to be described, but two* namely the Italian (It) 02 and QX viruses*appear to behave somewhat differently from our traditional view of IBV. It was soon clear that the variant It02 was widely distributed throughout many European countries, often being the most common IBV detected in surveys. However, associating it with disease outbreaks proved more difficult. The QX virus on the other hand, which is highly pathogenic, was first reported in China in the 1990s, then quickly spread across Russia and was reported in many European countries including The Netherlands, Germany, France and Belgium, and eventually England and Spain. Initially in China the virus was associated with proventriculitis, but in Europe it soon came recognized as a cause of nephritis in young birds and false layers in layers, breeders and backyard birds (see above). At the present time, QX continues to be a problem in many countries, and there is evidence that it has emerged in South America, although not in the USA. Meanwhile, in Australia a third group of IB variants was identified by Ignjatovic, represented by the ck/Australia/N1/03 and ck/Australia/N2/04 viruses. One of the more significant outcomes of the SARS coronavirus-related research during this decade was a more in-depth understanding of the rapid mutation rate characteristic of all coronaviruses including IBV, which can lead to the emergence of new virus types capable of causing disease. In the USA, the Arkansas-type virus was found to be persistent in broiler flocks, providing an opportunity for that virus to evolve and mutate to socalled Ark-like variants. In addition, evolution of the DE072 strain, leading to the origin of the GA98 strain of IBV, was reported. The GA98 variant virus became so widespread that a commercial vaccine was prepared for use in the USA. Vaccines. Research using biotechnology aimed at developing new IB vaccines was a very active area at this time. Molecular vaccines including subunit vaccines, DNA vaccines, virus-like particles and recombinant vaccine vectors have all been examined for their efficacy against IBV. In addition, an infectious clone against IBV, developed by Cavanaghs group, was extremely valuable for identifying pathogenicity-related areas in the IBV genome. However, only partial protection against homologous challenge has been reported. Despite the many advances in molecular biology, and our increased understanding of the virus, IB vaccines continue to be produced by technology that has been in use for over 50 years, namely passage of the virus in embryonated eggs. The main barriers to producing recombinant

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vaccines against IBV appear to be reproduction of the conformationally dependent epitopes on the spike glycoprotein that induce neutralizing antibodies and induce a protective local immune response in the upper respiratory tract of the bird. At a practical level, and despite experimental evidence from the UK and The Netherlands that a combination of existing vaccines can be efficacious against this variant, QX-type live vaccines have been developed in Europe with so far restricted use. This is probably a reflection of how strong the call from the field can be when it is felt that a new vaccine is needed!

and Heffels-Redmann, but nothing was decided. Maybe the Coronavirus Study Group of the International Committee for Taxonomy of Viruses could introduce a standardized nomenclature system for all coronaviruses, including IBV. Why do some infectious bronchitis variants have an (almost) worldwide distribution, whilst others remain localized? It is well known that some IBVs, such as Mass and 4/91, appear to have spread worldwide, while others including the major American types (apart from Mass), remain localized, but we do not know why this occurs. Despite increasing numbers of reports of IBV or IBV-like viruses in wild species there are no recognized wild bird reservoirs, which could transmit IBVs over long distances, such as the situation with AIVs and migratory waterfowl. We might suspect non-viral factors to include legal or illegal movement of stock or meat products. However, using the example of the QX variant coming from Asia to Europe, movement of 1-day-old stock would normally be in the opposite direction. Egg transmission can be eliminated, since although at a certain stage after infection there are high titres of virus in the oviduct, there are no reports of this phenomenon being blamed for new outbreaks. Viral factors might include survivability, virulence, or the mode of pathogenesis; possibly differences in duration of excretion from the respiratory and enteric tracts. However, in our present state of knowledge, we remain largely ignorant of these factors. Vaccines. Does the future lie with further advances in the molecular approaches that have been studied by various groups in recent years, with the hope of being able to tailor a specific vaccine to each new variant of importance? In the absence of a pan-IB vaccine, which seems a distant prospect, the use of a combination of two different vaccines providing broad heterologous protection seems to provide a good standby for the present. Molecular studies. Understanding the mechanisms behind the evolution of IBV and the emergence of new variants will be important for the future control of the disease. Currently research is being conducted at the Universities of Georgia and Auburn, USA on the selection of virus subpopulations in vivo, and the development of viral molecular diversity through mutations and recombination as the virus replicates in the host. This new avenue of research is contributing to our understanding of the role transmission plays in the antigenic and genetic drift and shift of the virus, and is an important step in preventing the emergence of new variants.

The future The disease and pathogenesis. Female reproductive tract. Apart from knowing that IBV replicating in the epithelium initiates effects in the oviduct, the detailed mechanism of how oviduct then ovary activity ceases and what initiates resumption of egg production has never been studied in detail. Since application of appropriate liveattenuated and inactivated IB vaccines can prevent egg production and quality loss, there seems likely to be little appetite for such investigations, even though IBV replication in the oviduct could act as a model system for understanding how viruses in other species can affect the reproductive processes. The kidney. An important epidemiological aspect of nephropathogenic IBVs is that they usually do not spread in the same way as some other IBVs, such as Mass and 4/91. For example, the European variant B1648 was responsible for outbreaks of kidney disease in a defined area of Northern France, The Netherlands and Belgium, but not elsewhere, even though isolated detections are made infrequently in other European surveys using PCR technology. The reason for this is not known. An important recent example of a nephropathogenic IBV is QX, and the kidney involvement continues to be an important manifestation in young birds in Europe. Puzzlingly, however, in China where the virus originated, it was associated with proventriculitis but not nephrosis. Long-term persistence of IBV in the chicken. Experimental data have shown that IBV can persist in the chicken and be re-excreted at point of lay, but the mechanism(s) involved and its significance in chicken flocks in the field are still poorly understood. It is possible that the tools are now available for this to be studied in more detail. Standardized nomenclature for infectious bronchitis variants. Although new IBV variants continue to emerge worldwide, seemingly in ever-increasing numbers, unlike the situation with Newcastle disease virus or AIV, there is no commonly used system for naming these variants. Despite the recent, genuine attempt in the literature to adapt nomenclature similar to that used for AIV, IBVs continue to be named according to the system used in the particular laboratory where they were first isolated. This is both cumbersome and confusing. For many years, the issue was raised at the series of Symposia on IB organized at Rauischholzhausen, Germany by Kaleta

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Summary This review has attempted to highlight the major achievements in IB research over the past four decades and to point to areas where knowledge is still lacking. Although the ciliated epithelial cells of the respiratory tract and oviduct and the tubular cells of the kidney have long been known to be the principle target cells for IBV, more recently the villus tips of the intestine were

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Swayne, D.E. (Eds). Diseases of Poultry 12th edn (pp. 117 135). Ames, IA: Blackwell Publishing. Cavanagh, D., Davis, P.J. & Mockett, A.P.A. (1988). Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralization epitopes. Virus Research, 11, 141 150. Cunningham, C.H. (1970). Avian infectious bronchitis. Advances in Veterinary Science and Comparative Medicine, 14, 105 148. de Wit, J.J. (2000). Technical Review. Detection of infectious bronchitis virus. Avian Pathology, 29, 71 93. de Wit, J.J., Cook, J.K.A. & van der Heijden, H.M. (2011). Infectious bronchitis virus variants: a review of the history, current situation and control measures. Avian Pathology, 40, 223 235. Dhinakar Raj, G. & Jones, R.C. (1997). Infectious bronchitis virus: immunopathogenesis of infection in the chicken. Avian Pathology, 26, 677 706. Fabricant, J. (1998). The early history of infectious bronchitis. Avian Diseases, 42, 648 650. Jackwood, M.W., Hilt, D. A., Lee, C-W., Kwon, H.M., Callison, S.A., Moore, K.M., Moscoso, H., Sellers, H. & Thayer, S. (2005). Data from 11 years of molecular typing infectious bronchitis virus eld isolates. Avian Diseases, 49, 614 618. Keeler, C.L.Jr., Reed, K.L., Nix, W.A. & Gelb, J.Jr. (1998). Serotype identication of avian infectious bronchitis virus by RT-PCR of the peplomer (S-1) gene. Avian Diseases, 42, 275 284. Kwon, H.M., Jackwood, M.W. & Gelb, J.Jr. (1993). Differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorhism analysis. Avian Diseases, 37, 194 202. Worthington, K.J., Currie, J.W. & Jones, R.C. (2008). A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006. Avian Pathology, 37, 247 257.

shown to be receptive to virus replication*but the significance of this is not clear. The most recent work confirms that the testis can be a site for IBV replication; a finding of potential importance for the transmission of IBV. The generalized distribution of IBV in the chicken and uncertainty regarding sites of possible virus persistence, together with the range of types of disease the virus can cause, all indicate that our knowledge of IBV pathogenesis and epidemiology is far from complete. Our understanding of immunity against IBV, particularly in regard to the role of cell-mediated responses, is lacking. The ability of the virus to change continually by mutation or recombination challenges our ability to both diagnose and control it. This suggests that 40 years of research into this virus and the disease has not been enough, and the areas outlined above indicate that many more years of research remain to be undertaken!

Further Reading
Bijlenga, G., Cook, J.K.A., Gelb, J.Jr. & de Wit, J.J. (2004). Development and use of the H strain of avian infectious bronchitis virus from the Netherlands as a vaccine: a review. Avian Pathology, 33, 550 557. Binns, M.M., Boursnell, M.E.G., Cavanagh, D., Pappin, D.J.C. & Brown, T.D.K. (1985). Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV. Journal of General Virology, 66, 719 726. Cavanagh, D. & Gelb, J.Jr. (2008). Infectious bronchitis. In Saif, Y.M., Fadly, A.M., Glisson, J.R., McDougald, L.R., Nolan, L.K. &

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