Emergence of subtype strains of the Arkansas serotype of infectious bronchitis virus in Delmarva broiler chickens

Infectious bronchitis virus (IBV) field isolates of the Arkansas (Ark) sero type were identified by reverse transcription-polymerase chain reaction (RT -PCR) as the most common serotype isolated from 1993 to 1997. These isolate s were recovered from broiler flocks with respiratory disease raised on the Delmarva peninsula in spite of Ark vaccination in the region. For the purp oses of investigating this apparently paradoxical finding, five RT-PCR Ark- positive held isolates recovered in 1995 and 1996 were selected for further characterization. The isolates were compared with Ak reference strains by reciprocal virus neutralization (VN) in embryonated eggs, S-1 gene sequence analysis, and challenge of immunity studies in specific-pathogen-free (SPF ) chickens. Antigenic (VN) comparisons and S-1 gene analysis confirmed that the five RT -PCR Ark-positive field isolates were of the Ark serotype but also revealed that the viruses could be readily distinguished from Ark reference strains . Four of the isolates (Ark/213/96, Ark/15C/96, Ark/1529/95, Ark/1534/95) w ere found to have higher antigenic relatedness percentages to each other (9 5%-100%) than to Ark reference strains DPI (52%-72%) and Georgia variant (G eorgia var) (53%-68%) by VN. Another isolate, Ark/1535/95, was found to dif fer antigenically from the other four RT-PCR Ark-positive field isolates (3 4%-61%), Ak DPI (44%), and Georgia var (43%) strains. The trends in the S-1 gene sequencing results were similar to those observed for the VN findings . Isolates Ark/213/96, Ark/15C/96, Ark/1529/95, and Ark/1534/95 demonstrate d a higher degree of predicted S-1 amino acid similarity to each other (96. 5%-98.7%) than to Ark DPI (92.4%-93.7%), Ark 99 (93.2%-94.7%), and Georgia var (89.3%-90.8%). Ark/1535/95 S-1 amino acid similarity values were lower compared with those of the other four RT-PCR Ark-positive field isolates (9 3.4%-94.8%), Ark DPI (91.9%), Ark 99 (93.0%), and Georgia var (88.7%). Furt hermore, the isolates could be distinguished from the Ark reference strains by a characteristic sequence polymorphism, a six-nucleotide deletion encod ing amino acids 57 (Asp) and 58 (Asp) in hypervariable region 1 of S-1. On the basis of the VN and sequencing findings, isolates Ark/213/96, Ark/15C/9 6, Ark/1529/95, and Ark/1534/95 were considered to be a single subtype of t he Ark serotype. The fifth isolate, Ark/1535/95, may constitute another sub type of the Ark serotype. Vaccination of SPF chickens with a high-titering commercially available liv e vaccine containing the Ark DPI strain provided solid protection (>90%) ag ainst challenge with the RT-PCR Ark-positive field isolates. Immunization o f SPF chickens with Ark/213/96 produced 100% protection against challenge w ith the homologous strain, as well as isolates Ark/1535/95 and Ark 99 but l ower levels of protection against Ark DPI (58%) and Georgia var (55%). Primers for RT-PCR were designed to distinguish between the Ark subtypes an d the Ark reference strains on the basis of the characteristic six-nucleoti de deletion identified in the S-1 gene of the Ark subtypes. Retrospective a nalysis of RT-PCR Ark-positive isolates found that the Ark subtypes existed as early as 1992 in Delmarva broilers and became prevalent by 1995. With R T-PCR, restriction fragment length polymorphism analysis, and DNA sequencin g techniques, the presence of Ark subtype viruses was demonstrated in two c ommercial Ark DPI strain vaccines and in our Ark DPI laboratory stocks that were the original source of the virus used for vaccine development. The de monstration of the Ark subtype and reference strains in the Ark DPI strain is evidence of the existence of IBV quasispecies. Factors possibly influenc ing the emergence of the Ak subtype in commercial broilers are discussed.