infectious bronchitis ( ib ) , caused by the infectious bronchitis virus ( ibv ) , is a highly contagious viral disease of chickens with a worldwide distribution .
it is characterized by respiratory symptoms , nephritis , poor performance and reduced egg reproduction . despite extensive vaccination
, ib has been a perpetual problem in the korean poultry industry since the disease was first reported in 1986 [ 7,9 - 11,16,17 ] .
recently , phylogenetic analysis revealed that korean field isolates of ibv constitute at least three distinct phylogenetic types : k - i , k - ii and k - iii .
k - i type strains are native viruses associated with localized incidence in korea , k - ii type strains are closely related to nephropathogenic ibv strains isolated in china , and k - iii type strains are closely related to enteric ibv variants of chinese strains . in particular , the k - ii type nephropathogenic ibv is associated with a high mortality rate , and has been the source of great economic losses in the poultry industry in korea , despite the availability of a vaccine based on the km91 strain of korean nephropathogenic ibv [ 7,9 - 11,17 ] . molecular epidemiological evidence suggests that k - ii type qx - like viruses ( k - iib type ) isolated since 2003 form a different cluster than those ( k - iia type ) isolated prior to 2003 ( e.g. , strain km91 ) .
nevertheless , antigenic differences between k - iia and k - iib type viruses have yet to be fully investigated .
the purpose of this study was to investigate the pathogenic and antigenic characteristics of k - iib type ibv strains currently circulating in korea to determine whether available ibv vaccines based on k - iia type strains will confer cross - protection against field ibv strains .
two korean ibv strains , km91 and kr / q43/06 ( national veterinary research and quarantine service , korea ) , were analyzed .
strain km91 is a nephropathogenic k - iia type virus that was isolated in early 1990s , and strain kr / q43/06 is a k - iib type virus isolated in 2006 .
the pathogenicity of ibv strain kr / q43/06 was determined using specific pathogen free ( spf ) 1-week - old layer type chickens ( lohmann , germany ) .
ten chickens were inoculated with ibv kr / q43/06 ( 10eid50/dose ) by eye drop inoculation .
five birds that served as the inoculation control group were inoculated with phosphate buffered saline .
all birds were maintained in isolators with negative pressure and monitored daily for 14 days for overt clinical symptoms ( depression , respiratory signs , diarrhea , etc . ) and mortality .
six days post inoculation ( dpi ) , all of the birds in the ibv group started to exhibit mild respiratory symptoms , including sneezing and rales .
clinical signs became more severe by 10 dpi , when most of the affected birds ( 7/10 ) exhibited severe depression and dyspnea , which continued to progress until the end of the experiment ( 14 dpi ) .
birds in the control group showed no clinical signs of ibv infection throughout the experimental period . in the infected birds , gross lesions
were mainly confined to the kidney , which was pale , swollen with interstitial nephritis and showed evidence of severe urate deposition .
ibv was recovered from the trachea ( 90% , or 9/10 ) and kidney ( 80% , or 8/10 ) of infected birds by inoculating tissue homogenate into the allantoic cavity of 9 to 11 day - old spf embryonated chicken eggs ( lohmann , germany ) using standard methods   and identified by dot - immunoblotting assay using monoclonal antibodies .
control birds exhibited no pathological lesions in the kidney , and no evidence of viral load .
cross virus neutralization tests were performed using anti - sera against strains km91 , kr / q43/06 and h120 to determine the antigenic relationship between kr / q43/06 and km91 .
ibv anti - sera were raised in our laboratory according to the immunization protocol of gelb et al .
cross - virus neutralization tests were performed using spf embryonated chicken eggs and the alpha method , as previously described . briefly , serial dilutions of ibv are incubated with a standard dilution of serum ( 1/5 dilution ) , and then the serum - virus mixtures in each dilution were incubated into 5 spf embryonated chicken eggs .
end - points were calculated by the reed and muench method . a neutralization index ( ni )
was calculated , representing the difference ( in logarithmic units ) in viral titer between virus alone and the corresponding virus - antiserum mixture .
an ni value of > 4.5 indicated homologous strains , a value of 1.5~4.5 indicated heterologous strains , and a value of < 1.5 was considered a different serotype .
as shown in table 1 , strain kr / q43/06 was effectively neutralized by antiserum to the homologous strain ( ni of 6.2 ) . however , the ni for antisera to strains km91 and h120 was 1.4 and 1.2 , respectively , which indicated that antisera to strains km91 and h120 were unable to effectively neutralize strain kr / q43/06 .
ni values were converted to antigenic relatedness ( r value ) using the following formula created by archetti and horsfall : where r1 is the ratio of the ni value of virus 2 in the presence of antiserum to virus 1 ( heterologous ni value ) to the ni value of virus 1 in the presence of antiserum to virus 1 ( homologous ni value ) ; r2 is the ratio of the heterologous ni value of virus 1 to the homologous ni value of virus 2 ; and r defines the antigenic relatedness of the two viruses . for assessing the similarity of the two viruses
, we used the criteria of brooksby , as follows : an r value of 100% or close to
100% indicated antigenic identity between the two tested viruses ; an r value > 70% indicated little or no difference between the two viruses tested ; an r value between 33% and 70% indicated a minor subtype difference ; an r value between 11% and 32% indicated a major subtype difference ; and an r value between 0% and 10% indicated different serotypes . the r value for strains kr / q43/06 and km91 was 30% , which indicated that nephropathogenic strain kr / q43/06 is distantly related to strain km91 ( table 2 ) .
thus , strain kr / q43/06 represents a new variant that is antigenically distinct from strain km91 . to our knowledge , this is the first report of a new antigenic variant of nephropathogenic ibv in chickens from korea . in a previous study
, phylogenetic analyses showed that type k - iib ibv isolates are more closely related to strain qx than the k - iia type strain km91 at the level of the hyper - variable region of the s1 gene of ibv .
it is worth noting that the qx - like ibv is distributed worldwide , including in the far east of russia , italy , the netherlands , the united kingdom and japan .
thus , k - iib type strains of ibv might have emerged in korea as a result of introduction from foreign countries rather than through immune pressure due to vaccination in the field , or recombination between field viruses .
interestingly , during viral surveillance in 2003 in quangdong , china , a nephropathogenic strain of ibv was isolated from an asymptomatic teal ( anas ) of the order anseriformes .
this observation raises the possibility of widespread dissemination of ibv by wild birds , such as wild migratory ducks , as proposed by bochkov et al .
while the role of wild birds in trans - boundary transmission of ibv has not been demonstrated , additional viral surveillance studies of wild birds , particularly wild ducks , are needed . in vivo
cross protection studies should also be considered to determine whether currently available k - iia type or mass type ibv vaccines confer cross - protection against new k - iib type variants .
if currently available ibv vaccines do not provide cross - protection against new variant strains that emerge in the field , the development of alternative vaccines should be considered for the effective control of ib in korea .
 despite the existence of an active vaccination program , recently emerged strains of nephropathogenic infectious bronchitis virus ( ibv ) in korea have caused significant economic losses in the poultry industry . in this study , we assessed the pathogenic and antigenic characteristics of a k - iib type field strain of ibv that emerged in korea since 2003 , such as kr / q43/06 . 
 specific pathogen free 1-week - old chickens exhibited severe respiratory symptoms ( dyspnea ) and nephropathogenic lesions ( swollen kidneys with nephritis and urate deposits ) following challenge with the recent ibv field strain . the antigenic relatedness ( r value ) , based on a calculated virus neutralization index , of the k - iib type field strain and k - iia type strain km91 ( isolated in 1991 ) was 30% , which indicated that the recent strain , kr / q43/06 , is a new variant that is antigenically distinct from strain km91 . 
 this report is the first to document the emergence of a new antigenic variant of nephropathogenic ibv in chicken from korea . 
Introduction