Being a coronavirus and, therefore, a single-stranded RNA virus, infectious c1 v1 c2 v2 virus (IBV) has an enormous capacity to change by spontaneous mutation and genetic recombination. It is believed that spontaneous mutations and genetic recombination can occur randomly in the IBV genome; however, if these events occur in the spike (S) gene, especially in its hypervariable regions, these events are most likely to result in the emergence of many different antigenic or genotypic types, which are commonly referred to as variants. Therefore, IBV is ubiquitous in most parts of the world where poultry are reared, and it is able to spread very rapidly in non-protected chicken flocks, leading to heavy economic losses in poultry industries (Cavanagh and Gelb, 2008). In general, while many new variants are unable to replicate or survive for a long time, a few variants that are of economic importance have emerged worldwide or in restricted geographic areas. Therefore, effective surveillance, which is primarily based on the isolation and identification of the virus type causing disease, is of great importance.
Classification or typing of IBV strains is very important for implementing control measures, research purposes, and understanding the epidemiology and evolution of IBVs. Thus far, two major groups of classification systems, including functional tests that examine the biological functions of a virus (immunotypes, protectotypes, and serotypes) and non-functional tests that assess the viral genome (genotypes), are commonly used for IBV typing (de Wit, 2000). For a specific IBV strain, although evidence from some studies suggests that there is a high correlation between the genotype and serotype, other studies have presented conflicting data (de Wit, 2000; Zhang et al., 2015; Chen et al., 2015), which may lead to contradictory results. The disadvantages of each system are that they only analyze one or several characteristics of a virus strain. Hence, data from only one system has to be interpreted with caution, while a more objective and accurate conclusion can be drawn by completely analyzing the results from different systems, although it is suggested that the preferred typing system usually depends on the goal (e.g., selection of vaccination programs or epidemiological studies), available techniques, experience, and costs (de Wit, 2000).
A large number of IBV genotypes and variants have been isolated in China in recent years (Han et al., 2011), among which two genotypes, the LX4 and ck/CH/LDL/97I types (also known as QX- and Q1-like, respectively), were first isolated in China and subsequently have become widespread worldwide (Valastro et al., 2016). Among these IBV genotypes, the TW1 type was first isolated in 1992 in Taiwan, and it was considered to have a different genotype than all of the other IBVs (Wang and Tsai, 1996). The majority of TW1 IBVs isolated from Taiwan are nephropathogenic, and they have mortality rates ranging from 10 to 60% in 1-day-old specific-pathogen-free (SPF) chickens (Wang and Tsai, 1996). However, the naturally recombinant TW I (nrTW I) genotype was first isolated in 2009 in China, and it was thought to have originated from a natural recombination between LX4 and TW1 viruses (Xu et al., 2016). Despite a previous study that characterized the genetic characteristics of an nrTW I type virus and its nephropathogenicity in 7-day-old SPF chickens (Xu et al., 2016), there is no further information on this important IBV variant. To better understand the nrTW I type, a series of experiments was performed to investigate its antigenicity and pathogenicity in the oviducts of SPF layers, and to evaluate the protection provided by commercial vaccines and attenuated viruses.
Materials and methods
In this study, most IB vaccines, including two types of government-approved vaccines (H120 and LDT3-A) and two types of non-government-approved vaccines (4/91 and Conn) that are used commonly in China, as well as the nrTW I type strains, were selected for genotyping, an S1 gene comparison, serotyping, and vaccination-challenge tests. In addition, we also selected four attenuated strains representing two types (the LX4 type strains LDL/091022, LSD/120720, and LGX/100508, and the ck/CH/LDL/97I type strain LDL/97I) of IBV strains that are believed to have originated in China and are the predominant IBV types circulating in chicken flocks in China and many other parts of the world (Valastro et al., 2016). A phylogenetic analysis and an S1 gene comparison showed that the aforementioned six types of viruses are genetically different from each other and from strain nrTW I. This is in accordance with the results of the cross-neutralization tests that showed that the serotype of strain nrTW I differs from those of the selected six serotypes in this study and likely represents a novel serotype. The cross-neutralization tests did not include the TW I prototype strain TW2575/98 from Taiwan, China, which was unavailable for the comparison.