Molecular characterization of variant infectious bronchitis virus in China, 2019: Implications for control programmes

Abstract Infectious bronchitis virus (IBV), an ongoing emergence enveloped virus with a single‐stranded positive‐sense RNA genome, belongs to the Gammacoronavirus genus in the Coronaviridae family. IBV‐associated tracheitis, nephritis, salpingitis, proventriculitis and egg drop have caused devastating economic losses to poultry industry worldwide. Since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with IBV in China. Here, we described two IB outbreaks with severe respiratory system or kidney injury in IBV‐vaccinated commercial poultry farms in central China. Other possible causative viral pathogens, including avian influenza virus (AIV), Newcastle disease virus (NDV) and Kedah fatal kidney syndrome virus (KFKSV), were excluded by reverse transcription‐polymerase chain reaction (RT‐PCR), and three virulent IBV strains, HeN‐1/China/2019, HeN‐2/China/2019 and HeN‐101/China/2019, were identified. Although the gross pathologic appearance of these two IB outbreaks was different, the newly identified IBV strains were all closely related to the ck/China/I0529/17 strain and grouped into GI‐19 genotype clade based on the sequencing and phylogenetic analysis of the complete S1 genes. Moreover, there are still some evolutionary distance between the newly identified IBV strains, HeN‐101/China/2019 in particular, and other GI‐19 strains, suggesting that Chinese IBV strains constantly emerge and evolve towards different directions. In conclusion, this study provided an insight of the recently emerging IBV outbreaks in IBV‐vaccinated commercial poultry farms and identified the genetic characteristics of three virulent GI‐19 IBV strains, which shows the need to carry out proper preventive measures and control strategies.


| INTRODUC TI ON
Avian infectious bronchitis (IB), a common avian disease with high morbidity and mortality, has drawn great attention for causing devastating economic losses to poultry industry worldwide (Jackwood & Wit, 2013). Infectious bronchitis virus (IBV), the causative agent, is an ongoing emergence enveloped, single-stranded, positive-sense RNA virus, with genome length about 27.6 kb. It is the prototype of the Gammacoronavirus genus in the Coronaviridae family (Bande et al., 2017;Masters & Perlman, 2013). IBV mainly targets the respiratory tract of its natural host-chicken, causing severe respiratory system disease, and spreads via respiratory and feco-oral routes. Additionally, IBV also infects the uro-genital tract, digestive system and reproductive system, resulting in nephritis, salpingitis, proventriculitis and egg drop (Bande et al., 2017;Jackwood & Wit, 2013;Sjaak de Wit, Cook, & van der Heijden, 2011).
A great number of IBV serotypes, genotypes and pathotypes have been identified worldwide since its first description in 1931 in America (Bande et al., 2017;Lin & Chen, 2017). To prevent IBV infection, researchers have developed various commercial inactivated and live attenuated vaccines over the past decades, which successfully prevented IBV infections (Jordan, 2017). As a highly mutable coronavirus, however, the continuous emergence of novel IBV strains greatly emasculate vaccines efficacy, and the low cross-protection rates of IBV vaccines inevitably hamper the prevention and control of the disease (Fan et al., 2018;Jordan, 2017).
Since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with IBV in China, especially in the intensive poultry raising regions, such as Henan, Hebei and Shandong provinces. Herein, we reported two IB outbreaks in IBV-vaccinated commercial broiler farms in central China, and the complete S1 genes of the newly identified IBV strains were sequenced, and its genotype, phylogeny and variations were analysed further. This study systematically described the genotype and evolutionary characteristics of the emerging IBV strains and highlighted the importance of continuous extensive surveillance to help choose suitable vaccines and develop control programmes reasonably.

| Broiler farm history and case presentation
Case 1: In April 2019, an acute outbreak of fatal respiratory disease occurred in a commercial broiler farm in Henan Province, Central China. According to the breeder's description, approximately onetenth of 20,000 25-day-old broilers, vaccinated with commercial live attenuated IBV vaccine H120 at 7-day-old, showed severe respiratory symptoms, such as gasping, nasal discharge, tracheal rales, sneezing, and some affected flocks also presented greenishyellow diarrhoea, watery eyes and lethargy. The outbreak started from 18 April 2019, and antibiotic-antimycotic combination therapy did not work. A total of ten dead broilers (5/10) and illness broilers (5/10) were selected randomly to send to the laboratory for diagnosis.
Case 2: In September 2019, an outbreak of fatal nephritis disease occurred in another commercial layer farm in Henan Province.
According to the breeder's description, the farm has approximate 9,000 layers with 21-day-old, which were vaccinated with commercial live attenuated IBV vaccine H120 at 7-day-old. The great majority of the flocks started to show moderate respiratory symptoms from 10 September 2019, and mitigated in a week with the antibiotic and traditional veterinary drugs combination therapies. Since 20 September, about 200 chickens per day died acutely without obvious clinical symptom. Six moribund layers were selected randomly to send to the laboratory for diagnosis.

| Ethical statement
The sick chicken examined in this study was approved by the poultry farm owners. Diagnosis and experimental protocols in this study were approved by the Henan University of Animal Husbandry and Economy Animal Care Committee. The clinical samples used in this study were collected in strict accordance with the guidelines for Animal Ethics Committees. All the chickens were euthanized by cervical dislocation and then examined the anatomical changes, samples of lungs, tracheas and kidneys were collected from sick chicken and stored at −80°C until used for diagnosis and virus isolation.

| Molecular diagnosis
The samples were homogenized and centrifuged to collect supernatants. Total viral RNA was extracted by using TRIzol (Invitrogen, Cat No: 15596026) according to the manufacturer's instructions, and further reversely transcribed to cDNA as described in our previous report . IBV, AIV, NDV and KFKSV (Palya et al., 2019), which can cause severe respiratory symptoms, nephritis and acute death, were analysed by RT-PCR using specific primers as previously described (Nguyen et al., 2013). Moreover, the positive samples were homogenated and then isolated in 9-day-old specific pathogen-free (SPF) eggs via the allantoic cavity, and the allantoic fluid was harvested sterilely and stored at −80°C.

| Gene amplification and sequencing
To identify the genotype of IBV strains identified in this outbreak, the complete S1 genes were amplified by using primers as follows: IBV-S1 forward: 5′-GAACAAAAGACCGACTTAGT-3′ and IBV-S1 reverse: 5′-TATGTACTCATCTGTRACAGT-3′, which were designed based on the conserved regions flanked the S1 gene of IBV strains downloaded from the ViPR database (http://www.viprb rc.org/brc/home.spg?decor ator=vipr). The correct size amplicons, with length about 2000 bp, were then cloned into pCloneEZ vector (Clone Smarter) and sequenced.

| Phylogenetic and recombination analysis
The complete S1 genes of the newly identified strains were subjected to online BLAST program (https ://blast.ncbi.nlm.nih.gov/Blast.cgi), and sequences sharing more than 95% nucleotide identify were downloaded for further genetic analysis. Additionally, the S1 genes of representative IBV strains within different genotypes and lineages Jiang et al., 2017;Ma et al., 2019;Valastro et al., 2016) were also downloaded for sequence alignment with Clustal Omega (https ://www.ebi.ac.uk/Tools/ msa/clust alo/), and the phylogenetic tree was conducted by MEGA 7.0 with the neighbour-joining method using 1,000 bootstrap replicates, which were then visualized by iTOL v4 software (Letunic & Bork, 2019). Recombination events among IBV strains were further investigated using the RDP4 software, a widely used tool for analysing individual recombination events and overall recombination patterns, by seven different algorithms recombination detection program (RDP), Bootscan, MaxChi, GENECONV, Chimaera, SiScan and 3Seq (Martin, Murrell, Khoosal, & Muhire, 2017).

| Visualization of mutations within the IBV S1 protein structure
Previous studies have revealed that most of the variable amino acids in S1 region were distributed in the three hypervariable regions (HVRs), including HVR I (38 aa-67 aa), HVR II (91 aa-141 aa) and HVR III (274 aa-387 aa), which were associated with neutralizing epitopes and receptor-binding domain and usually employed to classify IBV genotypes (Moore, Jackwood, & Hilt, 1997;Valastro et al., 2016). To further investigate the HVRs and mutations in the newly identified IBV strain, the 3D structure of HeN-2/China/2019 S1 protein was constructed by homology modelling method. From the PDB database, the glycoprotein S1 structure of vaccine M41 strain (Accession number: 6CV0) was downloaded and employed as the template for modelling S1 monomer (Shang et al., 2018). All the HVRs and mutations of HeN-2/China/2019 and HeN-101/China/2019 were located on the structure and visualized by PyMOL software (http://www.pymol.org/).

| Internal lesions
Among the organs collected from the morbid chicken among two IB outbreaks, the predominant histologic lesions were in the trachea and kidney, respectively. In case 1, a total of ten dead broilers (5/10) and illness broilers (5/10) were selected randomly to examine the anatomical changes, and trachea, especially between bronchus and bronchioles, showed the gross pathologic appearance of typical tracheitis, with serous, catarrhal or caseous exudate in trachea ( Figure 1a-c). Moreover, some of them presented systemic colibacillosis or airsacculitis, and no obvious nephritis and proventriculitis were observed. In case 2, all the six moribund layers exhibited severe lesion in kidneys, which were characterized by pale, swollen and mottled (Figure 1d,e). Some of them showed distended ureters filled with uric acid, but none of them presented tracheitis.

| Molecular diagnosis
To identify the causative agent of this outbreak, RT-PCR assays were used to detect the common potential viral pathogens, including IBV, NDV, AIV and KFKSV. As shown in Figure S1a

| Genetic and recombination analysis
To further characterize the biology and ecology of the newly identified IBV strains, we sequenced the complete S1 genes of Phylogenetic relationship among IBV strains has been established on the analysis of complete S1 gene. As shown in Figure 2, the phylogenetic tree further revealed that the HeN-1/China/2019, HeN-2/China/2019 and HeN-101/China/2019 strains were F I G U R E 2 Phylogenetic analysis of the complete S1 genes of newly identified IBV in 2019. Phylogenetic analysis based on the deducted amino acid sequences of complete S1 gene of HeN-1/China/2019, HeN-2/China/2019, HeN-101/China/2019 and other IBV isolates available from GenBank database. Phylogenetic tree was constructed by MEGA 7.0 software using the neighbour-joining method with 1,000 bootstrap replicates. The seven different IBV genotype, with 35 distinct lineage IBV strains were illustrated by the colour scale with iTOL software. Meanwhile, the available IBV vaccine strains in China were indicated by blue star, and the three IBV isolates in this study were indicated by red star [Colour figure can be viewed at wileyonlinelibrary.com] clustered together with ck/China/I0529/17, QX and LX4 strains and grouped into the GI-19 genotype, which were distantly from the available commercial vaccines H120, 4/91 and LDT3-A.
As a highly variable coronavirus, numerous IBV variants have been identified and nucleotide substitutions or recombination between field strains and vaccines occurred frequently. Previous study revealed that the recombinant events have already occurred in European strains within the S1 gene (Moreno et al., 2017). We further investigated the recombinant events between GI-19 and other IBV genotypes using the RDP4 software. The results showed that several recombinant events occurred between GI-19 and other IBV genotypes with high score (p < .01, recombinant score >0.6), including strains of the newly identified genotype GI-28, GI-25 and unassigned strain LDT3 ( Figure S2).
Interestingly, the GI-19 genotype plays a two-faced role in these recombinant events, not only providing fragments for other strains, but also receiving fragments during infection.

| Amino acid polymorphism analysis
To further explore the characteristics of the newly identified IBV strains, amino acid polymorphism of the S1 glycoprotein was compared with other IBV strains of all 35 genotypes Jiang et al., 2017;Ma et al., 2019;Valastro et al., 2016). The result revealed that most of the amino acid mutations in S1 region of the newly identified IBV strains were located in the three HVR regions.  (Table 1). Compared with the available capsid structure of M41 strain in the PDB database (Shang et al., 2018), the P87L and Q89K mutations are adjacent to HVR I and HVR II region structurally (Figure 3c) and located in the major receptor-binding domain (19 aa-253 aa; Promkuntod, van Eijndhoven, de Vrieze, Grone, & Verheije, 2014); the Q97E, S117A, T120R, A130E and R131S are in HVR II region; the T300I and H329Y are in HVR III region (Figure 3d), indicating that these mutations might change virus antigenicity.

| DISCUSS IONS
IB is an economically important avian disease that affects the poultry industries worldwide, particularly in the large poultry-producing countries, such as USA, China and Brazil (Bande et al., 2017;Jackwood & Wit, 2013). The first case of IB in China dates back to early 1980s, and since then, the outbreaks were constantly emerging and numerous IBV strains were identified, of which strains in genotype GI-19 (refers to the QX-like or LX4) became predominant among chickens currently, although the QX strain was initially identified in 1996. Additionally, other genotypes of IBV, such as GI-1 (Mass), GI-7 (TW-I or TW-like), GI-13 (4/91-like), GI-16 (Q1-like) and GI-22 (YN-like and SAIBK-like), were also frequently detected in China (Han et al., 2018;Li et al., 2012;Lin & Chen, 2017;Mo et al., 2013). Importantly, several novel IBV genotypes, including GI-28, GI-29 and GVII-1, were identified recently Jiang et al., 2017;Ma et al., 2019). All these indicated that the IBV strains circulating in China are genetically diverse, providing a potential platform for recombination and greatly challenging the current biological control measures.  (Bande et al., 2017;Han et al., 2011;Li et al., 2012). GI-1 genotype vaccine H120 has been the most widely used vaccine in China until now, which shared a very low homology of identity with the emerging GI-19 strain HeN-1/China/2019 (32.2%)

TA B L E 1
and HeN-2/China/2019 (32.2%) in glycoprotein S1 gene region, and even the other GI-19 strains identified recently. These results indicated the H120 vaccine could not provide effective protection against GI-19 genotype strains infection, which might explain the clinical phenomenon that a number of IBV outbreaks emerged in vaccinated flocks (Lin & Chen, 2017

F I G U R E 3
Characterization and localization of specific nonsynonymous mutations in capsid protein of the newly identified IBV strains in comparison with other IBV isolates. The multi-alignment of S1 glycoprotein of all IBV genotypes was conducted by Clustal-Omega, and schematic diagram based on the identified protein functional domains of mature S1 protein (without 1aa-19aa) was illustrated (a); 3D structure template of GI-1 genotype (M41 strain) was downloaded from PDB database, and the HVR regions of IBVs (b) and the location of mutation sites of HeN-2/China/2019 (c and d) were visualized by PyMOL software [Colour figure can be viewed at wileyonlinelibrary.com]