This article was downloaded by: [181.51.9.68]On: 12 December 2012, At: 05:03Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Avian PathologyPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/cavp20

The long view: 40 years of infectious bronchitisresearchJane K. A. Cook a , M. Jackwood b & R. C. Jones ca 138 Hartford Road Huntingdon, Cambridgeshire, PE29 1XQ, UKb Department of Population Health, College of Veterinary Medicine, University ofGeorgia, 953 College Station Road, Athens, GA, 30602, USAc School of Veterinary Science, University of Liverpool, Leahurst Campus, Neston, SouthWirral, CH64 7TE, UKAccepted author version posted online: 02 Apr 2012.Version of record first published: 18Jun 2012.

To cite this article: Jane K. A. Cook, M. Jackwood & R. C. Jones (2012): The long view: 40 years of infectious bronchitisresearch, Avian Pathology, 41:3, 239-250

To link to this article: http://dx.doi.org/10.1080/03079457.2012.680432

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss, actions,claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.

REVIEW

The long view: 40 years of infectious bronchitis research

Jane K. A. Cook1, M. Jackwood2* and R. C. Jones3

1138 Hartford Road Huntingdon, Cambridgeshire PE29 1XQ, UK, 2Department of Population Health, College ofVeterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA 30602, USA, and 3School ofVeterinary Science, University of Liverpool, Leahurst Campus, Neston, South Wirral CH64 7TE, UK

The remit of this review is to provide the non-specialist reader of Avian Pathology with an overview of researchcarried 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 firstidentified in the 1930s. Infectious bronchitis virus is a significant pathogen in the domestic chicken, affectingthe 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 foundonly in more local areas. This review mentions the major discoveries concerning both the virus itself and thetypes of disease it causes and considers recent changes in its pathogenesis. It also discusses the impact ofdevelopments in the field of molecular biology and highlights possible areas for future work.

Introduction

Infectious bronchitis (IB) is a highly contagious viraldisease of the chicken. It is possibly the most economicallyimportant viral respiratory disease of chickens in regionswhere there is no highly pathogenic avian influenza virus(AIV) or velogenic Newcastle disease virus and is foundeverywhere that commercial chickens are kept. Whileinitially a respiratory disease, the virus also affects thefemale reproductive tract, causing loss of production andpoor egg quality. Some strains have a predilection for thekidney of young chickens, resulting in nephritis that cancause significant mortality. Control is attempted using live-attenuated and inactivated vaccines. IB was first describedby Schalk and Hawn in the USA in 1931 as a respiratorydisease of chicks and the viral cause established in 1936.Infectious bronchitis virus (IBV) is a coronavirus; anenveloped virus containing a single-stranded positive-senseRNA and projections called spikes on the surface of thevirion that are involved in host cell attachment and induceneutralizing antibodies (Figure 1). The ability of IBV togenetically mutate and recombine results in antigenic shiftand drift. This means that vaccine programmes areconstantly being challenged. Thus, even though the diseasewas first reported more than 60 years ago, control is nevercomplete even today.

To celebrate the 40th anniversary of the journal AvianPathology, this paper provides an overview of the historyof research into IB, highlighting the major findings,assessing the current disease state and looking towardsfuture research. Because this is not intended as a formalreview we have not included full references since so manypeople have contributed to our knowledge of IB. Moredetails can be found in the list of further readingincluded at the end of this brief review. The authorsapologize to those colleagues whose work has not been

mentioned specifically but who have nonetheless made avaluable contribution to our knowledge of this interest-ing and challenging virus.

Infectious bronchitis research prior to first publication ofAvian Pathology

The disease and pathogenesis*general. Ciliated epithelialcells in the upper respiratory tract, epithelial cells in thefemale reproductive tract and tubular epithelial cells inthe kidney are the main targets for IBV. For several yearsthe virus has been tentatively linked with gut malfunc-tion, and more recently it has been suggested as a causeof male infertility and venereal spread of virus from maleto female.

Respiratory tract. Clinical signs include depression,snicking, coughing, head-shaking and nasal and oculardischarge. Necropsy findings include tracheitis, inflam-mation of the lungs and thickened and frequently cloudyair sacs. Birds that die usually suffer asphyxiation due tocaseous plugs in the bronchi resulting from secondarybacterial infection. In the 1940s and 1950s, histopatho-logical studies performed after experimental infection byHofstad in Iowa and by Jungherr and his colleagues atConnecticut demonstrated primary effects on the epithe-lial surfaces of the respiratory airways and frequentlyloss of ciliated epithelial cells accompanied by infiltra-tion of subepithelial layers with lymphocytes andheterophils. In the lungs, the disease affects the bronchiin a similar way to the trachea; diffuse infiltration beingthe only other change. Tissue recovery is normally seenafter 10 to 14 days.

*To whom correspondence should be addressed. Tel: �1 706 542547517. E-mail: mjackwoo@uga.edu

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

Received 25 March 2012

ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/12/030239-12 # 2012 Houghton Trust Ltdhttp://dx.doi.org/10.1080/03079457.2012.680432

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

Female reproductive tract. The role of IBV in adverselyaffecting egg production and quality has long beenknown. It became apparent in the 1950s that two periodsin the hen’s life are critical for the control of IBV: one ator after the point of lay and, less well appreciated, thesecond soon after hatching. The possibility that IBVcould affect egg production and quality during lay wasfirst studied by several American groups, includingDelaplane and Stuart, and Fabricant. In the 1950s,Van Roekel and his colleagues in Massachusetts, in a 10-year study of laying flocks, found that egg productiondrops varied widely, between 2 and 70% in affectedflocks, and eggs laid subsequently had weak, cracked ormisshapen shells with watery albumen. The likelihood ofother complicating pathogens cannot be ruled out, but ina systematic experimental study a few years later,Sevoian and Levine in Georgia, USA induced a fall inegg production similar to that seen in field cases. Theyshowed that the effects of the disease were moreprolonged than simple disease-induced loss of food orwater intake and that histological changes occurred inthe oviduct in particular, which influenced the nature ofeggs to be laid subsequently. Interestingly, IBV infectionduring the growing period appears to have little effect onthe ability of the hen to produce eggs of normal quality.As early as 1957, Urban and Goodwin reported thatinfection at 11 to 12 weeks of age did not influence laterproduction, while infection between 19 and 22 weeks ofage inflicted serious egg loss, and Box in the UK foundthat infection at 18 weeks of age caused a fall of 35% butat 6 weeks of age the subsequent loss was 13%. All ofthese reports relate to different field or experimentalconditions and virus strains, but the same pictureemerges.

At about this time, it became evident that with someIBV strains infection of female chicks soon after hatchcould also be influential. Broadfoot and his colleaguesstudied the performance of 35 layer flocks with sub-optimal production which all had respiratory diseasewithin the first 3 to 5 days of life. IB was diagnosed byserum neutralization tests and egg production wasreduced by up to 50%. Individual non-layers were foundto have abnormal oviducts that were non-patent andcystic, so that eggs could not be laid externally, although

the ovaries remained normal. Such birds have beentermed ‘‘false’’ or ‘‘silent’’ layers. The proportion of aflock affected depended on the age at infection, in that at1 day old the loss was 26%; at 18 days of age, 9.3%. Afew years later Crinion and Hofstad in Iowa and Jonesand Jordan in Liverpool, UK confirmed these findings.The American group found differences in effects betweenIBV types but could detect the development of oviductabnormalities in the form of cysts as soon as 6 weeksafter infection. Jones and Jordan showed that a patho-genic strain of the common Massachusetts (Mass) typecould cause these effects in chicks with no maternalantibodies and, by immunostaining, attributed thebizarre oviduct development to virus replication inoviduct epithelium soon after infection (Figure 2).

The kidney. From the 1950s, certain serotypes of IBVhave been described as nephropathogenic, causing con-siderable swelling of that organ, with urates in thecollecting tubules. This damage to the kidneys in youngbirds can result in significant mortality. The early workin this area was performed in the USA by Winterfield(Perdue University) and Hitchner (Cornell University),who described the Holte and Gray strains and used themto reproduce the disease in susceptible chickens. How-ever, Cumming and his colleagues in Australia, workingwith the Australian T strain, did much of the pioneeringwork in this area. Kidney damage was the prominentclinical manifestation of Australian IBVs in the 1960s.Cumming showed that factors which predisposed chick-ens to the condition include chicken genetic line, dietaryprotein source, as well as chilling and other stress factors,and speculated that nephritis is a condition that perhapsall IBV strains are capable of inducing, given therequired circumstances. In part, this was substantiatedby the report of Jones in the next decade who describedmortality due to nephritis and demonstrated virus inkidneys by immunostaining, in chicks accidentallychilled after infection with a not normally nephropatho-genic 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�100 nm

Figure 2. Section of oviduct from a female chick 3 days after

intranasal inoculation with Mass-type IBV. Immunofluorescence

stain shows virus in the epithelial cells. Bar �50 mm.

240 J. K. A. Cook et al.

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

Diagnosis of infectious bronchitis. IBV affects chickens ofall ages, involving the respiratory, renal and reproductivesystems, and because the clinical signs are not specificthe need for differential diagnostic methods was quicklyrealized. These methods focus on either detecting orisolating the virus itself or detecting serum antibodies toit. Pioneering work in the USA by Cunningham (1970)at East Lansing and by Fabricant (1998) at CornellUniversity established that IBV grows well in embryo-nated eggs, and passage of field material via the allantoicroute of 9-day-old embryos became the method of choicefor virus isolation. The virus causes characteristic curlingand stunting of the embryo, visible after about 7 daysincubation and, after serial passage, embryo mortalitymay be observed with field strains. This method ofdiagnosis continued to be used for many years although,because of the need to rely on embryo stunting as anindication of IBV infection, and the frequent need forserial passage of field material before typical effects areseen, interpretation of the results was always subjective.Furthermore, the method is time consuming (7 daysincubation being necessary before embryo lesions areobserved) and expensive, requiring a significant numberof embryos. However, passage in embryonating eggs isstill in use to propagate the virus today.

These issues made serological diagnosis seem a moreattractive option, but again this was not simple in thecase of IBV. In the early 1950s Fabricant developedstandardized procedures for IB diagnosis and for inter-pretation of the findings using the serum neutralizationtest in embryonated eggs. In those days, the test wasnormally done using a single serum dilution testedagainst a series of dilutions of the virus, which addedto the cumbersome nature of the test. In the early 1960s,Woernle in Germany pioneered the use of an agar gelprecipitation test for IBV. Although used for somedecades, probably because it is cheap and easy toperform, this test proved to be very insensitive andstandardization of the test between different laboratorieswas never achieved. Its only merit seems to be that,because of the transient nature of precipitins, a positiveresult gives some indication of a recent infection.

Whatever the diagnostic approach taken, there havealways been difficulties to overcome because the virusaffects so many tissues, making the choice of material forIBV isolation or detection critical. Since the primarytarget tissue for IBV is the upper respiratory tract,trachea or nasal tissue is the material of choice wheninvestigating respiratory disease. However, the viruspersists in the respiratory tract for a relatively shorttime after infection, and therefore kidney, or intestinaltract swabs or tissue, are the materials of choice wheninvestigating kidney infection or poor egg laying perfor-mance. Interestingly, the reproductive tract has neverbeen a productive tissue for isolation of IBV, perhapsbecause of the transient presence of virus there. ClearlyIB diagnosis was an area in need of improvements andthese were to come in the next decades.

The immune response to IBV. The cross-neutralizationtest developed by Fabricant (see above) enabled the levelof humoral antibodies post IBV infection to be detectedand quantified. The test also allowed distinction betweenstrains in terms of the degree of cross-neutralizationusing antisera against each strain. However, Raggi’steam at Davis in California was the first to demonstrate

a lack of correlation between titres of circulatingantibodies and resistance to infection.

Infectious bronchitis variants. The first realization thatIBV was not homogeneous came with the observation byJungherr in 1956 that an IBV isolated in Connecticut didnot cross-protect chickens against challenge with theoriginal Mass isolate. This led to the awareness of theexistence of more than one IBV serotype or variant asthey were to be called and had important implicationsfor control. For many years this was thought to representthe first emergence of an IBV different from the Masstype. However, with the benefit of both hindsight andmodern technology, we now know as a result of work byNaqi’s group at Cornell University that non-Mass-typeIBVs have been present in the USA from the early 1940sand many more were subsequently reported.

Vaccination against IBV. Attempts to control IBV werereported as early as the 1940s when Van Roekel showedit was possible to protect birds against the seriouseconomic effects of IBV infection during the layingperiod by exposing them during rear to IBV that hadbeen attenuated by passage in embryos. This was a veryearly example of the use of ‘‘controlled exposure’’ toprotect against IBV and involved infecting a few birdswith the virus and letting it spread naturally through therest of the flock; a crude method but nonethelesseffective. This approach also provided maternal anti-bodies to progeny, which were found to have someprotective value initially. These observations gave im-petus to the development of vaccination procedures.Early on, it was recognized that live-attenuated IBvaccines could be developed by reducing or attenuatingthe virulence of the virus by serial passage in embryo-nated eggs. Because IBV will only grow in cell cultureonce it has been adapted to do so by serial passages inthe laboratory, the procedure of attenuating IBV byembryo passage, in use from the 1950s, is still employedtoday.

In the early 1950s, the first IB vaccine was developedin the USA using the van Roekel M41 (Mass) strain. Bythe early 1960s IB had been diagnosed in The Nether-lands, and this led to the development, by the Dutchveterinarian Bijlenga et al. (2004), of a vaccine based onan IBV isolated in that country and known as the Hstrain of the Mass serotype. The letter ‘‘H’’ stands for thename of the farmer (Huyben) from whose chickensthe isolate was made and not, as is often thought, for thecountry, Holland. The resulting vaccines, known asH120 and H52, soon became widely used and theH120 vaccine is arguably still the most widely used IBvaccine worldwide today. The names give an indicationof what is involved in attenuating an IB virus; H120 is amild vaccine used from as young as 1 day of age and hadreceived 120 embryo passages, whilst H52, for use onlyas a second vaccine, had received only 52 passages.

Infectious bronchitis research 1970 to 1979

The first publications on IB in Avian Pathology were byJordan and Nassar, looking at the survival of IBV inwater, and by Hitchner, who examined the limitations ofthe virus neutralization (VN) test for classifying fieldisolates*both papers were published in Volume 2 (April

Forty years of IB research 241

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

1973). Subsequent publications during the 1970s ad-dressed all aspects of the disease as well as diagnosis andcontrol.

The disease and pathogenesis. Renewed interest in theeffects of IBV on the female reproductive tract wasshown in this decade. Darbyshire and Cook, working atHoughton Poultry Research Station (HPRS) in the UK,found that co-infection with either Mycoplasmagallisepticum or adenovirus worsened the effects. Apartial explanation for how IBV affects the oviductwas provided by Jones and Jordan, who demonstratedwith immunostaining that after respiratory infectionIBV replicates in all regions of the oviduct epithelium,and in the case of the pouch shell gland, to very hightitre (Figure 3). The Liverpool work demonstrated thatthe effects on egg production of individuals variedgreatly after identical respiratory application of thevirus, with some birds out of lay for 3 to 4 days whileothers experienced a pause of more than 70 days. Thisgroup found substantial amounts of virus in the oviductafter IBV infections of hens, which posed the question ofwhether the virus is egg transmitted. In this decade, bothMcFerran at Stormont, Northern Ireland and Cook atHPRS found evidence of this when virus was isolatedfrom chicks hatched from infected hens. However, inview of the significant reduction in the number of eggslaid by infected chickens and no reports of spontaneousoutbreaks of IB in isolated flocks, this phenomenon, if ithappens, seems likely to be of little importance.

The intestine. For the first time, interest was shown in thepossible role of the intestinal tract in IBV infections.Several papers in the 1970s by Alexander and hiscolleagues at the Central Veterinary Laboratory(CVL), Weybridge, UK and by Cook mentioned excre-tion of IBV strains in the faeces after cloacal swabbing,sometimes associated with apparent long-term persis-tence of virus in individuals or in the flock. Thesignificance of enteric excretion has never been fullydetermined, except that it contaminates the environ-ment, but the survival of IBV in faeces is poorlyunderstood. Over the subsequent decades, various

reports described isolation of IBV from the intestinesof experimentally infected chickens; and in the 1990s,Jones and colleagues confirmed this using immunostain-ing to show virus in villus tips at different regions in thegut, but with no apparent effect on gut function.

Diagnosis of infectious bronchitis. With the identificationof new IB variants came the need for improveddiagnostic methods to identify them, but developmentscontinued to be hampered by the need to grow IBV inembryonated eggs. Progress came in the 1970s after IBVhad been adapted to grow in cell culture, and Hopkins inGeorgia, USA was able to show that plaque-purifiedIBVs could be compared in that much simpler system.Hopkins’ work enabled both field isolates and seracollected from disease outbreaks to be studied in plaquereduction tests in cell culture. At about the same time,Johnson and Marquardt in Maryland developed thetechnique of performing VN tests in tracheal organcultures using a constant amount of virus and varyingdilutions of serum. This led to improved, quicker andcheaper methods for IB diagnosis and for differentiatingIB variants.

Following the observation that some IBV strains,when treated with the enzyme phospholipase C, wereable to haemagglutinate chicken erythrocytes, Alexanderand his colleagues developed a standardized procedurefor performing the haemagglutination inhibition (HI)test for IB serology. Standardization was found to bevery important because of the variable introduced by theneed to enzyme-treat IBV before use. The test becamewidely used in IB diagnosis, although Bracewell andBrown at CVL demonstrated its limitations for differ-entiating between variants due to the high level of cross-reactions found between them. The HI test continues tobe used and limited differentiation of variants may bepossible provided good controls are included andexperienced technicians perform it.

Immune response to IBV. The 1970s saw the publicationof several papers beginning to investigate the role ofantibodies in the immune response to IBV. The avail-ability of the HI test enabled Gough and Alexander atCVL to confirm the lack of correlation between anti-body levels and resistance to disease in the respiratorytract. However, humoral antibodies were shown tocorrelate well with absence of virus recovery from thekidney and genital tract (i.e. remote tissues). Theimportance of B cells in IBV infections was studied byChubb in Australia, by depletion experiments usingtestosterone or cyclophosphamide. B-cell-depleted chick-ens showed increased clinical signs and more severelesions, especially in the kidney.

Infectious bronchitis variants. By the early 1970s, severaldifferent IB variants had been described in the USA,mainly using serology, and awareness of the problemsthat different IBVs could cause was beginning to berecognized. At that time almost all of the work in thisarea was being done by groups in America, includingJohnson and Marquardt, Hitchner, Hofstad, and Win-terfield, and important variants identified includedFlorida, Clark 333 and Arkansas. IB was reported forthe first time in South America in the mid-1970s, and inboth Brazil and Chile during this decade. Work done by

Figure 3. Section of magnum of a mature hen 6 days after

intranasal inoculation with M41 Mass-type IBV. Immunofluor-

escence stain shows virus in the epithelial cells. Bar �25 mm.

242 J. K. A. Cook et al.

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

Lohr demonstrated that IB variants were certainlypresent in Malaysia in the 1970s. There was thusincreasing awareness of different types of IB, and inthe 1970s Cunningham, one of the pioneers of IBresearch, was the first person to attempt to group IBvariants according to the immune response they induced.

Vaccines. By the early 1970s, vaccination against IB waswidely practiced and the live-attenuated vaccines H120and H52 were almost universally used worldwide, exceptin the USA where Mass-type vaccines based on the M41strain were developed. Importantly, Davelaar and Kou-wenhoven at the Doorn Institute in The Netherlandsshowed that maternally derived antibodies appeared tohave no adverse effect on the efficacy of live-attenuatedIBV vaccines given at 1 day of age. However, the inabilityof Mass-type vaccines to protect against all of the newIB variants that by now were emerging gave impetus tomoves to develop vaccines to some of these new variants.It will be clear from what has been said elsewhere in thisreview that the number of new IB variants beingdescribed was too large for a new vaccine to bedeveloped against each one. Furthermore, it was soonrealized that many of the variant viruses identified incommercial poultry disappeared so quickly that by thetime a new vaccine had been produced and tested, theproblem strain had gone away and been replaced by anew one. The skill was therefore in deciding whichvariant was of sufficient importance to warrant a specificvaccine. Vaccines developed in the USA at that timeincluded Florida, Arkansas, and Connaught & Holland(both Mass types).

Infectious bronchitis research 1980 to 1989

Long-term persistence of IBV in the chicken. Although inthe 1970s Alexander’s and Cook’s groups had reportedon the shedding of IBV in faeces for many weeks afterexperimental infection, a true latency, such as occurswith infectious laryngotracheitis herpesvirus residing inthe trigeminal ganglia, was never confirmed. However, inthe 1980s work at Liverpool showed that after infectionof 1-day-old female chicks, while virus could not bedetected during the growing period, one strain of IBVwas re-excreted sporadically at point of lay via both therespiratory tract and the gut. This occurred in thepresence of high titres of circulating antibodies, butwithout clinical disease. Hormonal activity at sexualmaturity was thought to be responsible for virusrecrudescence but oestrogen and progesterone injections,designed to induce early sexual maturity, failed to bringforward virus excretion before 20 weeks of age.

Diagnosis of infectious bronchitis. At the beginning of the1980s the serum neutralization test was widely used fordetecting and differentiating IB variants in serum, sincethis 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 andtime consuming. A big step forward therefore came withthe introduction of enzyme-linked immunosorbent as-says (ELISAs), which were initially developed for IBV byMarquardt and co-workers and proved to be highlysensitive, allowing early detection of IBV infections andneeding only small volumes of sera. A number of

commercial ELISA kits soon became available andcontinue to be widely used for IB diagnosis. These testsgive a large amount of information very quickly and areinvaluable for flock profiling and for monitoring re-sponses to vaccination. However, they do have theirlimitations. The large amount of data rapidly generatedmeans that careful interpretation of results is crucial andit is important to remember that ELISAs detect group-specific rather than type-specific antibodies. Nonethe-less, ELISAs continue to be the method of choice forserological monitoring for IBV.

Another big advance in this decade came with theproduction of monoclonal antibodies (mAbs) for use inIB research, and these soon found many applications.For example, Koch and his colleagues at Lelystad in TheNetherlands prepared a series of mAbs to IBV, whichwere specific for different structural proteins of the virus.On the basis of their reactivity to a panel of mAbsspecific for the spike protein, clear differentiationbetween IBV field strains was possible, providing amuch quicker method for this purpose than neutraliza-tion tests. However, a major limitation of the use ofmAbs for the differentiation of IBVs is that a smallmutation in the virus can lead to an amino acid changein the epitope with which a specific mAb reacts, leadingto a misdiagnosis.

Immune response to IBV. The work of MacDonald andcolleagues in Edinburgh confirmed the protective effectof antibodies on remote tissues such as the oviduct, andthis was echoed by Box who showed that IBV-specificantibodies were important in preventing drops in eggproduction due to IBV, by protecting the oviduct fromviraemic virus.

Vaccination studies have almost always focused onhumoral immune responses in relation to protection.Nevertheless, Darbyshire at HPRS illustrated lack ofcorrelation between antibodies and resistance, anddiscrepancies between in vitro strain differentiation byVN tests and in vivo cross-protection results. Further-more, it was shown by Jones and Ambali that, following1-day-old infection, re-excretion of virus at onset of laycan occur in the presence of high titres of circulatingantibodies. All this suggested that while antibodies playa role in recovery from IB, other immunologicalmechanisms are involved.

Immunity studies were further advanced during thisdecade, and possibly for the first time the importance oflocal and cell-mediated immunity in responses to IBVinfections was realized. That the S1 (spike) glycoproteinof IBV induces VN and HI antibodies was established byCavanagh et al. (1988) at HPRS and by Niesters atLelystad, and this was considered the most probableinducer of protection at this time.

The role of different classes of immunoglobulin (Ig)was also recognized in this decade. The major circulatingimmunoglobulin detected by HI was found to be IgG,and sensitive ELISAs were developed to measure it.Mockett and Darbyshire showed that anti-IBV IgGcould be detected as soon as 4 days post infection,reaching a peak at 21 days and remaining at high titre inserum for many weeks, making this a useful measure ofIBV infection and vaccine application. In contrast, anti-IBV IgM was shown by Mockett to be present onlytransitorily, reaching a peak at about 8 days and thendeclining. This finding was later proven to be a useful

Forty years of IB research 243

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

marker for recent infection, and ELISAs were developedto detect specific IBV IgM responses.

Studies on local immunity centred on investigating thepresence of IgA in lachrymal fluid, and the work ofDavelaar’s group showed the importance of the Hard-erian gland. IgA is particularly important in responses to1-day-old vaccination, and removal of the gland causeda decrease in protection. IgG could also be found intears but was mainly serum derived. Mockett reportedthat maternal antibodies could be detected in trachealwashes but these were shown to be short lived.

The first report of a possible role for cell-mediatedimmunity to IBV was by Timms and colleagues at CVL.They demonstrated antigen-specific proliferation of Tlymphocytes in IBV-infected chickens. It was also shownusing mouse mAbs that CD4 and CD8 antigens arefound on T-helper and T-cytotoxic/suppressor T cells,respectively. In Australia, Chubb demonstrated thepresence of cytotoxic lymphocytes in the spleen andperipheral blood.

At this time the only cytokine studied with regard toIBV was interferon gamma. Otsuki’s group in Japandetected variable levels, mainly in respiratory organs, butthe UK workers Holmes and Darbyshire could notdetect it. Interestingly, there seemed to be little interestin determining what role, if any, interferon has inresponse to IBV infections, until very recently whenCollison’s group in Texas addressed the interferongamma question and concluded that both natural andrecombinant forms delay disease onset and decreaseseverity.

Infectious bronchitis variants. Largely as a result of thework done by Kouwenhoven and Davelaar at Doorn,different IBVs suddenly became a big issue in Europe. Anumber of different variants (each given the prefix ‘‘D’’)were described associated with disease problems invaccinated flocks in The Netherlands and neighbouringcountries. Shortly afterwards, Cook and Darbyshiredeveloped the use of tracheal organ cultures as theirpreferred system, rather than embryonated eggs, forisolating and growing IBV and for performing cross-neutralization tests. They subsequently isolated manydifferent IB variants in the UK and other countriesworldwide; many of which were not controlled byexisting IB vaccines. This really was the decade of newIB variants. Over the next few years, similar as well asdifferent variants were being isolated throughout Europeand this led to renewed interest in improving controlmeasures to combat them. That this was a worldwideproblem became clear in the 1980s with reports of IBvariants from all continents and probably all countrieswith a developed poultry industry. In the USA, Hofstad,Gelb Jr (University of Delaware) and King (SoutheastPoultry Research Laboratory, USDA) were examiningthe cross-immunity of numerous IBV isolates. One of themost significant discoveries was the isolation of theArkansas-DPI virus from broilers on the DelmarvaPeninsula; this virus became the parent strain for all ofthe Ark-DPI-type vaccines currently used in the USA.By the end of the 1980s, researchers were becomingaware that the incidence and distribution of IB variantswas complex. Some viruses, such as Mass, occurredworldwide, the Arkansas virus appeared to be restrictedto certain regions of the USA, whereas others such asD274 were found in Europe but not in the USA. The

occurrence of new IB variants and their incidence anddistribution around the world is still unpredictable andconfusing today. For further information, see the reviewby de Wit et al. (2011).

Vaccines. Live-attenuated infectious bronchitis vaccines.Up to this time, most IB vaccines were based on theMass type. Interestingly, during the 1980s it becameclear in the USA that H120 vaccines from differentsources provided different levels of immunity and had anunusual ability to cross-protect against some of theheterologous IBVs found there, including JMK andFlorida. However, the emergence of an increasingnumber of variants led to calls for new IB vaccines tocombat them. It has long been the case within thepoultry industry that, if disease occurs in a flock, theblame is laid at the door of the vaccine itself, rather thanconsideration being given to whether or not that vaccinehas been used correctly! However, it soon became clearthat, whilst Mass-type vaccines could protect againstsome of the new types of IBV being found in both theUSA and Europe, this protection was not complete andnew vaccines were required to combat many of the newvariants now being reported. During this decade,vaccines such as D274 and D1466 were developed inEurope and used there and elsewhere, in situations wherethe prevalence of a new variant has been established.Problems encountered in the USA at that time involvedthe Arkansas variant, which was established as a majorproblem in many of the poultry-producing areas.Inactivated infectious bronchitis vaccines. By this decade,it was realized that live-attenuated IB vaccines were notproviding adequate protection throughout the life oflayers and breeders, and furthermore could sometimesaffect laying performance. This led to the developmentof inactivated vaccines for use in layers and breeders.Slow release of antigen and long lasting immunitythroughout the laying period are achieved by intramus-cular or subcutaneous injection of vaccines that incor-porate an adjuvant together with IBV that has beeninactivated by a method that destroys virus infectivitybut maintains antigenic structure. Unfortunately, inacti-vated IB vaccines are not effective if used alone, so birdsmust still be given one or a series of vaccinations withlive-attenuated IB vaccines during rear (live priming)prior to administering the inactivated vaccine. Othercrucial problems had to be overcome in the developmentof inactivated vaccines, namely finding the optimalmethod of destroying the infectivity of the virus withoutlosing its antigenicity and selecting the most suitable andeffective adjuvant. Both of these issues caused continu-ing problems to researchers and vaccine manufacturersalike, being reflected in the high cost of these vaccines.Nevertheless, inactivated vaccines appear to be cost-effective and continue to be used.

Beginnings of research on molecular characterization ofthe virus. This decade saw the first interest in studyingthe structure of the virus itself. Structural characteriza-tion of the spike glycoprotein and nucleic acid sequenceanalysis of the viral RNA was being conducted by Binnset al. (1985) at HPRS and by Niesters and co-workers atLelystad. The most significant finding was that neutra-lizing epitopes on the spike glycoprotein were encodedby hypervariable regions in the gene. These data laid the

244 J. K. A. Cook et al.

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

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

Infectious bronchitis research 1990 to 1999

Long-term persistence of IBV in the chicken. The site ofpersistence of virus has always been controversial, andBhattacharjee and Dhinakar Raj working with Jones atLiverpool carried out work in this area. One series ofexperiments pointed to the kidneys as the probable siteof persistence. After 1-day-old infection of chicks andlong after clinical disease and virus excretion had ceased,virus could be reactivated by the T-cell immunosuppres-sor cyclosporin. In serial tissue examinations aftertreatment, virus first reappeared in the kidney, then inrespiratory tissues, but was not detected in the caecaltonsil, considered by several workers to be the survivalsite. Dhinakar Raj argued that the kidney is animmunoprivileged site, where virus could survive awayfrom the immune response.

Diagnosis of infectious bronchitis. mAbs played a majorrole in the development of diagnostic techniques for IBVin this decade. Studies had begun on detection ofdifferent antibody classes in the response of the chickento IBV and the importance of IgM in the early immuneresponse to IBV infections was further recognised. Up tothis time, different classes of antibody could be detectedby affinity chromatography, but the availability of mAbsspecific to IgM led to the development by da SilvaMartins, then working at HPRS, of an IgM-specificELISA for IBV. This technology was developed furtherby Naqi and by de Wit and his colleagues at Deventer,who devised an IgM-antibody capture ELISA specificfor IBV. A different application of mAbs in IB diagnosisis illustrated by the work of Ignjatovic in Melbourne,Australia, who used mAbs directed against the threemain structural proteins of IBV to detect and differ-entiate the IB variants found in Australia.

Perhaps the most significant discovery of this decadewas that molecular characterization of the spike genecorrelated with serotype of the virus, which led to thedevelopment of molecular techniques to type IBV. In1991 Lin and co-workers at the Nippon Institute inTokyo, described the use of reverse transcriptase poly-merase chain reaction (RT-PCR) to amplify a 400-base-pair region of the spike, followed by restriction enzymefragment length polymorphism (RFLP) as a typingmethod for IBV. Shortly afterwards, Jackwood et al.(2005) at the University of Georgia in Athens, USAdescribed a similar procedure and showed that it couldbe routinely applied to field samples for rapid typing ofIBV isolates. That assay employed primers that amplifythe S1 gene of any IBV, followed by the use of threerestriction enzymes to produce a unique type-specificpattern of DNA fragments on an agarose gel. Thetechnique could be used for all IBVs. In addition,Jackwood’s group discovered that phenol-inactivatedviruses could also be typed. This very important findingallows IBVs from all over the world to be transported foridentification. A different approach was described byKeeler et al. (1998), at the University of Delaware, wherespecific primers were designed and used to identify anddifferentiate six different serotypes of IBV. As nucleicacid sequencing became easier and less expensive, RFLP

analysis gave way to sequence analysis of the RT-PCR-amplified spike genes. This was a vast improvement overRFLP analysis because sequences of the spike gene canbe aligned and used to compare the relatedness betweenknown strains and new variants (Figure 4). In addition,an explosion of sequence data for IBV spike genes fromall over the world was being submitted to GenBank,allowing anyone with access to the Internet to comparethe IBV types detected worldwide.

Immune response to IBV. Further studies showing theimportance of humoral antibodies in IBV infection byCook’s group using surgical bursectomy rendered anIBV-resistant chicken line (line C) more sensitive toinfection, with an increased severity and duration ofclinical disease. However, mortality rates did not in-crease, although humoral antibodies seemed to protectthe tracheal epithelium following secondary challenge.

Further work on local antibodies in the trachea,showing better correlation between lachrymal antibodiesand protection, led Toro’s group at Auburn, Alabama torecommend that lachrymal antibodies could be used forantibody profiling of flocks. The same group later foundthat a light layer breed had a higher serum IgG andlachrymal IgA response than a heavy brown layer breed.Cook’s groups meanwhile confirmed that chicken linesresistant to IBV had higher levels of IgA in tears thansensitive lines, although antibody titres in trachealwashes were similar. Local immunity in the oviductwas confirmed by Dhinakar Raj & Jones (1997), whodetected virus-specific IgG and IgA in oviduct washes ofIB-infected hens. In addition to local production, anti-bodies later transuded into the oviduct from the serum,but their value in oviduct protection remains to bedetermined. Using in vitro challenge of oviduct organcultures from vaccinated chickens, the same group foundin young chicks that local antibodies in the oviduct wereless protective than those in the trachea.

Regarding the intestinal tract, replication of IBV in thegut was earlier established by Lucio and Fabricant andby Ambali and Jones at Liverpool. Dhinakar Raj &Jones (1997) further demonstrated local antibody pro-duction in the duodenum and caecal tonsils of hensinfected with an enterotropic IBV strain. Previously,Lutticken and others in the Netherlands failed to detectantibodies in gut washings after H120 and H52 vaccineapplication, but the differences may have been due to theuse of different virus strains. The role of these antibodiesin limiting gut replication needs further elucidation.

Cell-mediated immunity to IBV was further exploredin this decade. Janse and co-workers at Lelystadcontended that immunity to IBV in the trachea ismediated by T cells. CD4 and CD8 cells were shown intracheal sections by this group and by Dhinakar Raj &Jones (1997). However, while the Dutch group found anincrease in CD4 cells, the UK workers found higherlevels of CD8 cells. Again, these differences may havebeen related to virus strain. The Liverpool group alsoshowed that after T-cell immunosuppression with cy-closporin, virus titres in kidneys were higher than inuntreated birds, suggesting that T cells may also protectthe kidney.

Infectious bronchitis variants. With the development ofmolecular-based diagnostic typing tests, identification of

Forty years of IB research 245

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

a new IB variant became possible in days rather thanweeks. Consequently, new genetic types were rapidlybeing identified worldwide, including in the USA,Central and South America, Europe, Australia and,particularly, in Japan, China, Taiwan and Korea. Someof the viruses from the Far East were shown to be closelyrelated to ones already reported from Europe and theUSA, but others appeared to be unique to particularcountries and even to specific regions of that country. Itwas soon apparent that the situation was extremelycomplex and difficult to interpret. Were new variantssuddenly emerging with much greater frequency, or wasit simply that we were able to type many more viruses ina short period of time? We do not know for sure, butwhat we do know is that many of these variants, whilstreadily detectable, do not seem to be of major signifi-cance in terms of the disease they caused, nor do theyappear to persist for very long.

Over the past 40 years, many IB variants have comeand gone, but just a few have haunted the poultryindustry for a long time. The Arkansas-type viruscontinues to be a problem in the USA and a cousin ofthat virus, the California variant first identified in 1991,still circulates in chickens there. In addition, the Dela-ware virus (DE072) identified in 1992 is still found incommercial poultry in the USA. Interestingly, theArkansas, California and Delaware viruses have notbeen reported in countries other than the USA. How-ever, this is not the case with the variant known variouslyas 793B; 4/91; CR88 (referred to here as 4/91). Thisvariant, first reported in the UK and France at thebeginning of this decade, is now found in many parts ofthe world, except, interestingly, the USA. The pathologycaused by 4/91 was not typical of other IBV infectionssince it was originally associated with mortality inbreeders, scouring in broilers and possibly musclemyopathy under field conditions. This variant continuesto be a problem in many parts of the world, but it doesnot appear to have changed greatly in its structure or inits antigenicity, such that the vaccines which wereoriginally developed to combat it still provide adequatecontrol today.

In Australia, several unique IBV types were identifiedwhich were only found in that country and were differentfrom the nephropathogenic Australia T strain identifiedin 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 IBvariant identified in China was the QX variant. Un-known at the time, this virus eventually became perhapsthe most significant IBV type apart from Mass in theyears to come (see below).

Vaccines. By the 1990s, the vast increase in the numberof IB variants being identified led to major concernsabout how best to protect against them. Developing anew vaccine against the multitude of new variantsidentified was clearly not possible. In addition, it takesseveral years to develop and licence a new vaccine, whichin hindsight is probably a blessing since many newvariants tend to be transient problems, coming andgoing in only a few months or years. During the 1990sCook, building on the findings of Lohr, who showed thatit was possible to group IBVs into ‘‘protectotypes’’,demonstrated that much broader protection could beobtained than that suggested by serotyping or genotyp-F

igu

re4

.P

hylo

gen

etic

tree

ba

sed

on

the

am

ino

aci

dse

qu

ence

so

fse

lect

edst

rain

so

fIB

V.

Th

en

eig

hb

ou

r-jo

inin

ga

nd

Nei�G

ojo

bo

rim

eth

od

sw

ere

use

dto

com

pu

teth

ere

late

dn

ess.

Th

ea

min

oa

cid

seq

uen

ces

wer

e

ali

gn

edw

ith

Clu

sta

lWfo

rIB

Vty

pes

:C

ali

forn

ia�

CA

V/C

AV

56

b/9

1;

Au

stra

lia

(su

bg

rou

p1

)�

N1

/62

S1

;G

eorg

ia0

8�

GA

08

/HS

p1

6/0

8S

1;

Ark

an

sas�

Ark

/Ark

DP

I/8

1;

D2

74

(E

uro

pea

ng

rou

pB

)�

B/D

27

4/

84

;D

38

69

(E

uro

pea

ng

rou

pE

)�

E/D

38

69

/84

;4

/91�

79

3B

/4-9

1/9

1;

Geo

rgia

07�

GA

07

/GA

07

/07

S1

;Q

X�

QX

/QX

IBV

/99

;M

ass

ach

use

tts�

Ma

ss/H

52

/S1

,M

ass

/H1

20

/S1

,Ma

ss/M

ass

41

/41

S1

;

Co

nn

ecti

cut�

Co

nn

/Co

nn

46

/51

S1

vacc

ine;

Flo

rid

a�

FL

/FL

18

28

8/7

1;

Del

aw

are�

DE

/DE

07

2/9

2S

1va

ccin

e;G

eorg

ia9

8�

GA

98

/04

70

/98

S1

;D

14

66�

Du

tch

/D1

46

6/8

1.

246 J. K. A. Cook et al.

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

ing. Based on analysis of ciliostasis in the trachea,Cook’s work showed that, using a vaccination pro-gramme including a vaccine of the Mass serotype andone developed against the 4/91 variant, it was possible toprotect against many, if not all, of the IB variantscausing problems in many parts of the world. It isinteresting to speculate whether this finding holds truefor other different combinations of IB vaccines, orwhether this is a property unique to these two particularstrains. Certainly they are very different from each othergenetically and are also both known to be strongimmunogens. The precise mechanisms of this phenom-enon have not been elucidated and this is an area thatmerits further research.

Infectious bronchitis research 2000 to 2011

Molecular biology. In 2002 the outbreak of severe acuterespiratory syndrome (SARS) caused by a coronavirus inhumans was significant for IBV research because a largenumber of laboratories and considerable resources wereimmediately targeted to research on coronaviruses.Many molecular characteristics of the virus were studied,including transcription and replication, protein synthesisand structure, virion assembly, mutation rates, recombi-nation and viral genetic diversity, infectivity and epide-miology.

The disease and pathogenesis. Tissue tropism. Abdel-Moneim and co-workers in Egypt showed by immuno-histochemical staining that, following inoculation ofembryos with the M41 strain, IBV could be detected inthe nasal epithelium, trachea, lung, spleen, myocardialvasculature, liver, gastrointestinal tract, kidney, skin,sclera of the eye, spinal cord, as well as in brain neurons.These results were consistent with virus isolation anddenote the wide tissue tropism of M41 in the chickenembryo, perhaps reflecting the relatively undifferentiatedstate of the embryonic cells, rather than confirming analmost pan-tropism for chicken tissues, which has notbeen demonstrated in the hatched chick.

The importance of Australian IBV research in thisdecade is demonstrated by the work of Ignatovic and hercolleagues, who interestingly showed in a 20-year studyof Australian IBVs that, over time, the kidney tropism,historically a consistent characteristic of IBVs in Aus-tralia, had changed to one for the respiratory tract andthat this had been due to a small sequence change in theS1 gene.

Effect of IBV on the female reproductive tract. In the lastdecade, notable work by Chousalkar and colleagues inAustralia has shown detailed differences between strainsof IBV in pathogenicity for the oviduct, including thefinding, using real-time RT-PCR, that some strains canpersist 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 the1970s, was largely forgotten since it had been seen rarelyover the last few decades, but it emerged again in thisdecade with a vengeance following the appearance of theQX genotype of IBV in Europe at the beginning of thelast decade. Although this variant was first reported inChina associated with proventriculitis, when it arrived inEurope it was found to cause nephritis in young birds

and silent layers in flocks in production, apparentlyresulting from infection at a very young age. This resultsin cystic lesions and, although the ovaries remainnormal, eggs are not laid due to the severe andpermanent damage to the oviduct. These effects havebeen reproduced experimentally by Benyeda and hiscolleagues in Hungary and by de Wit in The Nether-lands. The rarity of occurrence of this form of IBsuggests that not all IBVs have the same predilectionfor the oviduct, or perhaps that heterologous maternalantibodies cannot always neutralize viral infection in thefirst few days of life.

Male reproductive tract. Although Cook in the 1970sdetected IBV in the semen of infected cockerels, afinding that suggested venereal transmission could occur,this has been largely ignored until recently, when Bolzand co-workers in Illinois and Villarreal and hercolleagues in Brazil detected IBV in the testes ofcockerels in flocks where male infertility was suspected.Toro and co-workers recently confirmed the earlierfinding of Cook and demonstrated IBV in the testis byimmunostaining. Whether all IBVs have a tropism forthe cells in the testis that produce spermatozoa remainsto be determined, as does the importance of this finding.

Long-term persistence of IBV in the chicken. Early in thisdecade Naqi’s group showed that IBV could persist andbe shed in the trachea and cloaca of chickens for as longas 77 days. More recently, Jackwood’s group hasprovided evidence in favour of the caecal tonsil beingthe site of IBV persistence, by detecting virus in thistissue using immunostaining several weeks after infec-tion. Sufficient inconsistencies exist in the literature toindicate that perhaps the site of IBV persistence in thebird depends on the infecting virus strain and the age ofthe bird at infection. In small experimental groups it isusual to find that infectious virus is no longer detected afew weeks after infection. However, in commercial flocksof many thousands of birds, recycling of virus*a so-called rolling reaction*is likely to occur. Raj & Jones(1997) considered that long-term persistence might allowvirus mutation, but work by Naqi’s group suggests that,for the Mass strain at least, it does not result in changesin the nucleotide sequence of the S1 gene or in tissuetropism. The mechanisms and frequency of long-termpersistence in the epidemiology of IBV are unknown.However, it may have significance in that a clinicallynormal flock re-excreting virus could act as an unex-pected source of infection.

Diagnosis of infectious bronchitis. By this time, molecularbiological techniques were becoming widely used indiagnosis. This technology enabled large-scale epidemio-logical surveys to be conducted elucidating the globaldistribution of IBV variants. The finding that driedswabs and, a few years later, FTA cards impregnatedwith material that inactivates viruses but still permits thedetection of viral RNA by RT-PCR, provided a meansto examine an enormous number of samples from allover the world. Thus, large amounts of spike genesequence data quickly became available in GenBank.These data proved invaluable for comparing viruses, butit is critical to remember that in a diagnostic setting thetechnique detects viral RNA and gives no indication of

Forty years of IB research 247

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

whether or not live virus is present in the bird at the timeof sampling. This is an important epidemiological pointwhen trying to correlate the results of RT-PCR surveyswith the disease situation in the field. Furthermore, thetechniques do not provide live virus, which may beneeded for vaccination and challenge studies.

Immune response to IBV. This decade saw little majorwork on the more basic aspects of immunity to IBV.However, there are a few studies that warrant mention.Secondary bacterial infection following an initial IBVinfection has always been a major concern, and Verveldeand colleagues in Utrecht, The Netherlands examinedcolibacillosis and asserted that enhanced colibacillosisafter IBV infection or vaccination is caused at least byaltered innate immunity and less by impairment ofphagocytic cell function. The importance of IBV-specificmaternal antibodies was illustrated by De Herdt and hiscolleagues in Ghent, Belgium, who showed that eco-nomic losses associated with IB in broilers were foundpredominantly where maternal antibodies were low anderratic.

The interaction of IBV with other important patho-gens in the chicken has always been a concern, and theimmunosuppressive effects of chicken anaemia virus andinfectious bursal disease viruses on local antibodies toIBV infection were clearly shown by Toro’s group. Bothviruses caused a reduction in IBV-specific IgA in therespiratory tract, thereby confirming the major role thatimmunosuppressive agents may play in the chicken’sresponse to infection with IBV.

Cell-mediated immunity. This was further studied byCollisson and her colleagues in this decade. They showedthat memory T cells can be detected in blood for at least10 weeks after IBV infection, while virus-specific CD8memory cells can protect syngeneic chicks from acuteIBV infection. Further work on in vitro stimulation withIBV antigen showed that B cells can be activated tosecrete antibody by 3 weeks post infection. It wasasserted that detection of such activated cells could beuseful in early detection of infection.

Wang’s group in South Dakota followed the genetranscription profile of tracheal epithelial cells three daysafter infection of chickens with an attenuated IBV Massstrain. They confirmed that a diversity of innateimmunity and helper T-cell-type-1-biased adaptive im-munity are activated in the host’s early defence againstIBV invasion and are responsible for the rapid clearanceof virus from the local infection.

Innate immunity and other factors. In the past decade,several novel, sophisticated techniques have been appliedto research into IBV and have increased our knowledgeof this virus and of immune responses to it. They haveincluded aspects of innate immunity, immune modula-tors and genetic resistance. Some examples of thesepublications are mentioned here: Niu and co-workers inDalian, China reported that the innate response to IBVcould be stimulated by injection of baculovirus, whichenhanced inflammatory cytokine mRNA expression inneonatal chicks. Similar to vaccination developments inother species, the use of immune-modulating agents hasbeen reported. Babiuk and colleagues in Saskatchewanshowed that CpG oligodeoxynucleotide is able to limit

IBV propagation in embryonic tissues, and they suggestthat it might play a part in future vaccine design. Withregard to genetics of the chicken, Etches and colleaguesin East Lancing reported that the genes tightly linked tothe B haplotype are those relevant to IBV resistance.Finally, Wang’s group, in a further report on thecomplexity of immunity to IBV, contended that anumber of innate immune factors of mucosal immunityare activated after IB immunization, including Toll-likereceptors, type 1 interferons, interleukin 1 beta, comple-ment, T-cell signalling molecules, surface markers andeffector molecules.

Infectious bronchitis variants. During this decade, manynew variants continued to be described, but two*namely the Italian (It) 02 and QX viruses*appear tobehave somewhat differently from our traditional view ofIBV. It was soon clear that the variant It02 was widelydistributed throughout many European countries, oftenbeing the most common IBV detected in surveys.However, associating it with disease outbreaks provedmore difficult. The QX virus on the other hand, which ishighly pathogenic, was first reported in China in the1990s, then quickly spread across Russia and wasreported in many European countries including TheNetherlands, Germany, France and Belgium, and even-tually England and Spain. Initially in China the viruswas associated with proventriculitis, but in Europe itsoon came recognized as a cause of nephritis in youngbirds and ‘‘false layers’’ in layers, breeders and backyardbirds (see above). At the present time, QX continues tobe a problem in many countries, and there is evidencethat it has emerged in South America, although not inthe USA. Meanwhile, in Australia a third group of IBvariants was identified by Ignjatovic, represented by theck/Australia/N1/03 and ck/Australia/N2/04 viruses.

One of the more significant outcomes of the SARScoronavirus-related research during this decade was amore in-depth understanding of the rapid mutation ratecharacteristic of all coronaviruses including IBV, whichcan lead to the emergence of new virus types capable ofcausing disease. In the USA, the Arkansas-type viruswas found to be persistent in broiler flocks, providing anopportunity for that virus to evolve and mutate to so-called Ark-like variants. In addition, evolution of theDE072 strain, leading to the origin of the GA98 strain ofIBV, was reported. The GA98 variant virus became sowidespread that a commercial vaccine was prepared foruse in the USA.

Vaccines. Research using biotechnology aimed at devel-oping new IB vaccines was a very active area at this time.Molecular vaccines including subunit vaccines, DNAvaccines, virus-like particles and recombinant vaccinevectors have all been examined for their efficacy againstIBV. In addition, an infectious clone against IBV,developed by Cavanagh’s group, was extremely valuablefor identifying pathogenicity-related areas in the IBVgenome. However, only partial protection against homo-logous challenge has been reported. Despite the manyadvances in molecular biology, and our increased under-standing of the virus, IB vaccines continue to beproduced by technology that has been in use for over50 years, namely passage of the virus in embryonatedeggs. The main barriers to producing recombinant

248 J. K. A. Cook et al.

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

vaccines against IBV appear to be reproduction of theconformationally dependent epitopes on the spike gly-coprotein that induce neutralizing antibodies and inducea protective local immune response in the upperrespiratory tract of the bird. At a practical level, anddespite experimental evidence from the UK and TheNetherlands that a combination of existing vaccines canbe efficacious against this variant, QX-type live vaccineshave been developed in Europe with so far restricted use.This is probably a reflection of how strong the call fromthe field can be when it is felt that a new vaccine isneeded!

The future

The disease and pathogenesis. Female reproductive tract.Apart from knowing that IBV replicating in the epithe-lium initiates effects in the oviduct, the detailed mechan-ism of how oviduct then ovary activity ceases and whatinitiates resumption of egg production has never beenstudied in detail. Since application of appropriate live-attenuated and inactivated IB vaccines can prevent eggproduction and quality loss, there seems likely to be littleappetite for such investigations, even though IBVreplication in the oviduct could act as a model systemfor understanding how viruses in other species can affectthe reproductive processes.

The kidney. An important epidemiological aspect ofnephropathogenic IBVs is that they usually do notspread in the same way as some other IBVs, such asMass and 4/91. For example, the European variantB1648 was responsible for outbreaks of kidney diseasein a defined area of Northern France, The Netherlandsand Belgium, but not elsewhere, even though isolateddetections are made infrequently in other Europeansurveys using PCR technology. The reason for this isnot known. An important recent example of a nephro-pathogenic IBV is QX, and the kidney involvementcontinues to be an important manifestation in youngbirds in Europe. Puzzlingly, however, in China where thevirus originated, it was associated with proventriculitisbut not nephrosis.

Long-term persistence of IBV in the chicken. Experi-mental data have shown that IBV can persist in thechicken and be re-excreted at point of lay, but themechanism(s) involved and its significance in chickenflocks in the field are still poorly understood. It ispossible that the tools are now available for this to bestudied in more detail.

Standardized nomenclature for infectious bronchitisvariants. Although new IBV variants continue to emergeworldwide, seemingly in ever-increasing numbers, unlikethe situation with Newcastle disease virus or AIV, thereis no commonly used system for naming these variants.Despite the recent, genuine attempt in the literature toadapt nomenclature similar to that used for AIV, IBVscontinue to be named according to the system used inthe particular laboratory where they were first isolated.This is both cumbersome and confusing. For manyyears, the issue was raised at the series of Symposia onIB organized at Rauischholzhausen, Germany by Kaleta

and Heffels-Redmann, but nothing was decided. Maybethe Coronavirus Study Group of the InternationalCommittee for Taxonomy of Viruses could introduce astandardized nomenclature system for all coronaviruses,including IBV.

Why do some infectious bronchitis variants have an(almost) worldwide distribution, whilst others remainlocalized? It is well known that some IBVs, such asMass and 4/91, appear to have spread worldwide, whileothers including the major American types (apart fromMass), remain localized, but we do not know why thisoccurs. Despite increasing numbers of reports of IBV orIBV-like viruses in wild species there are no recognizedwild bird reservoirs, which could transmit IBVs overlong distances, such as the situation with AIVs andmigratory waterfowl. We might suspect non-viral factorsto include legal or illegal movement of stock or meatproducts. However, using the example of the QX variantcoming from Asia to Europe, movement of 1-day-oldstock would normally be in the opposite direction. Eggtransmission can be eliminated, since although at acertain stage after infection there are high titres of virusin the oviduct, there are no reports of this phenomenonbeing blamed for new outbreaks. Viral factors mightinclude survivability, virulence, or the mode of patho-genesis; possibly differences in duration of excretionfrom the respiratory and enteric tracts. However, in ourpresent state of knowledge, we remain largely ignorantof these factors.

Vaccines. Does the future lie with further advances inthe molecular approaches that have been studied byvarious groups in recent years, with the hope of beingable to tailor a specific vaccine to each new variant ofimportance? In the absence of a pan-IB vaccine, whichseems a distant prospect, the use of a combination oftwo different vaccines providing broad heterologousprotection seems to provide a good standby for thepresent.

Molecular studies. Understanding the mechanisms be-hind the evolution of IBV and the emergence of newvariants will be important for the future control of thedisease. Currently research is being conducted at theUniversities of Georgia and Auburn, USA onthe selection of virus subpopulations in vivo, and thedevelopment of viral molecular diversity through muta-tions and recombination as the virus replicates in thehost. This new avenue of research is contributing to ourunderstanding of the role transmission plays in theantigenic and genetic drift and shift of the virus, and isan important step in preventing the emergence of newvariants.

Summary

This review has attempted to highlight the majorachievements in IB research over the past four decadesand to point to areas where knowledge is still lacking.Although the ciliated epithelial cells of the respiratorytract and oviduct and the tubular cells of the kidneyhave long been known to be the principle target cells forIBV, more recently the villus tips of the intestine were

Forty years of IB research 249

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12

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 persis-

tence, 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, particu-

larly 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). Develop-

ment 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. &

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 field

isolates. Avian Diseases, 49, 614�618.

Keeler, C.L.Jr., Reed, K.L., Nix, W.A. & Gelb, J.Jr. (1998). Serotype

identification 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 bron-

chitis virus genotypes in Western Europe from 2002 to 2006. Avian

Pathology, 37, 247�257.

250 J. K. A. Cook et al.

Dow

nloa

ded

by [

181.

51.9

.68]

at 0

5:03

12

Dec

embe

r 20

12