Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in China during 2008-2009

Infectious bronchitis (IB) is a serious and highly contagious disease of chickens, accompanied by decreased egg production and poor egg quality in laying flocks. Avian infectious bronchitis virus (IBV) was first reported in the USA, replicating in the respiratory tract and some epithelial cells of gut, kidney, and oviduct [1‐3]. IBV commonly predisposed the birds to secondary infection with some bacterium, such as Escherichia coli and Mycoplasma gallisepticum, resulting in complicated disease process and increased mortality [4, 5]. The clinical disease and production problems frequently cause catastrophic economic losses to the poultry industry all over the world. IBV belongs to the genus Coronaviridae, family Coronaviridae, order Nidovirales, and possesses a single stranded positive-sense RNA genome encoding four structure proteins, phosphorylated nucleocapsid (N) protein, small envelope protein (E), integral membrane glycoprotein (M), and spike glycoprotein (S) [6, 7]. The S glycoprotein on the outside of the virus contains epitopes associated with serotype differences, and is cleaved post-translationally by cellular proteases into the S1 and S2 subunits [8, 9]. The globular S1 subunit forms the tip of a spike, extending outward, plays a role in attachment and entry into the host cell, which has relation to induce virus neutralizing antibody and hemagglutination inhibition antibody, whereas the S2 subunit anchors the S1 moiety to the viral membrane [8‐11]. Coding for the heavily glycosylated spike glycoprotein, the error-prone nature of RNA polymerase made the S1 gene could easily generate nucleotide insertions, deletions, point mutations, and RNA recombination under vaccine pressure, to bring about new variation strain and change of tissue tropism [12‐16]. It is documented that only a few amino acid differences amongst S proteins are sufficient to have a detrimental impact on cross-protection [15, 17‐20]. Antigenically different serotypes and newly emerged variants of field chicken flocks lead to vaccine breaks [21, 22].

Recently, more than 20 serotypes within IBV have been identified worldwide. The complex epidemiology characterize of IB raised the control difficulty. In China, since IBV strains were first isolated and identified in 1982, various live-attenuated and inactivated vaccines derived from Massachusetts (Mass) serotype strains have been widely and extensively used in chicken farms to reduce the adverse effect of the IBV [23, 24]. However, the disease continues to emerge and cause serious production problems, even occurred in routinely vaccinated layer and breeder flocks in China, and the situation gets worse as time progressed [25].

It was documented that nephropathogenic type IB has become more and more prevalent in China. The unprecedented economic losses caused by the nephropathogenic IB suggested that selecting the appropriate vaccine strain against the IB outbreaks is of great importance [25, 26]. However, the integrated natures of novel circulating IBV strains in mainland China were not well-learned.

The previous study by other researchers has been revealed that the variation in S1 sequences was closely confirmed relative to the emergence of novel strains, and S1 gene sequence was a good predictor of challenge of immunity in chickens [17, 18, 27]. This study was conducted to identify the IBV strains that have escaped immune defenses conferred by vaccination in China. The genetic characterization of recent IBV field isolates in China was performed by sequencing the whole S1 genes, sequence alignment and phylogenetic analysis compared with other reference strains.

Infectious bronchitis (IB) is one of the most common and difficult-control poultry diseases in China, caused persistent but infrequent outbreaks in commercial chicken farms [24, 25, 28]. Commercial vaccines based on H120, H52, 28/86, Ma5, W93 and M41 strains, have been widely used to control the disease [2, 29]. Natural outbreaks of IBV often are the result of infections with strains that differ serologically from the vaccine strains. Come to the rapid and complicated evolutionary of IBV, it is imperative to learn profoundly the circulating IBVs, facilitate selecting the candidate vaccine strain against the infections [2, 24].

In this study, 80 IBV strains were isolated from the vaccinated chicken flocks, with a wide age range of IB outbreak. The chickens infected before the age of 5 days which might be caused by the vertical transmission of IBVs or the maternal antibody could not provide pertinent protection against the prevalent strains [30]. Furthermore, there was accumulating evidence indicated that the nephropathogenic IBVs have become prevalent in China in last several years [23, 26, 31]. Through clinical records and the virus recovery trials, 70 identified isolates mainly caused typical swollen kidney, different from the respiratory type strains isolated in earlier years, including the major vaccine strains. These findings indicated that all 80 isolated IBV strains from China during 2008-2009 were evolutionarily distant from the vaccine strains used for current, resulting in vaccination failure cases.

The S1 protein determined the serotypic evolution, the phenotype change and the genetic diversity of IBVs [32]. In the present study, nucleotide and derived amino acid sequences of S1 protein genes of the 80 field strains were aligned and compared to the representative strains, to determine the relationship of circulating field isolates, vaccine strains and previously described variant strains. Newly isolated strains shared between 75.4% to 100% nucleotide sequence similarity with each other, higher similarity than the vaccine strains and other representative IBVs. Although the IBVs all over the world shared some common antigenic types, virus strains within a geographic region were unique and distinct, even in different provinces of China. The variants were mostly located in the first 300 amino acids in the N-terminal of the S1 protein of IBV, even though the mutants consisted of insertions, deletions and point mutations were complicated and detailedly different, the hypervariable regions in S1 protein in this study were similar to previous studies [19, 23, 26].

The phylogenetic analysis showed that there were five subgroups of IBVs co-circulating in China, and multiple strains might cause the constant IB outbreaks. The newly isolated strains were mostly derived from A2, 4/91 and HN08. Only CK/CH/Chongqing/0908 belonged to the branch of Gray. The phylogenetic distributions were closely relative to geographical factors. Most of the recently isolated IBVs in this study formed the distinct cluster related to the A2 type. However, the routine vaccine strains mainly belong to M41-type branch. A2 strain is closely related to 4/91 serotype, spreading over Europe since its first isolation in UK in 1991 [9, 24, 33‐37]. In this study, 61.3% (49/80) field isolates belonged to the A2-type branch, which included 85.7% (42/49) nephropathogenic field isolates of this study. The QXIBV, first isolated in China and reported associated predominantly with various forms of renal pathology in China, was also representative A2-type strain [25, 31]. The analysis results were according to the prevalence of nephropathogenicity IB. To date, the QX-like IBV strains have been widely isolated in many European countries, and become a dominant genotype [5, 38]. Through IB surveys, the European QX-like IBV strains have been reported that caused 86% respiratory signs, 22% litter or enteric problems, only 2% had swollen kidneys [39]. Absorbingly, the QX-like IBV strains have undergone divergent evolution paths, brought out different variants in Europe and China. Similarly, seven exceptional strains located in the A2-type branch caused evident respiratory problems, including three isolates from Zhejiang province (QZ1, QZ2 and QZ3) and three isolates from Guangdong province (XD2, XD3 and LZ2), and GL from Guangxi province. The results of our study indicated the strain grouping, such as phenotype and genotype, were not only depended on the geographical factors. The evolutionary pace and the epidemiology characteristics of the IBV were complicated.

In conclusion, the data obtained from our study suggest most of present IBV isolates in China are A2-like nephropathogenic strains. To control the prevalence and well prepare for the potential outbreaks of IB, the candidate virus strain for vaccination might be selected timely and specifically in a geographical region, which manifests the importance of continuing surveillance of new IBV strains. This paper is a periodic report on our ongoing surveillance program. We hope the study could contribute to guiding the development of effective vaccines and establishment of control policy for IB.