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Novel variants of infectious bursal disease virus can severely damage the bursa of fabricius of immunized chickens
Journal Pre-proof Novel Variants of Infectious Bursal Disease Virus Can Severely Damage the Bursa of Fabricius of Immunized Chickens Linjin Fan, Tiantian Wu, Yulong Wang, Altaf Hussain, Nan Jiang, Li Gao, Kai Li, Yulong Gao, Changjun Liu, Hongyu Cui, Qing Pan, Yanping Zhang, Xiaomei Wang, Xiaole Qi
PII:
S0378-1135(19)31053-3
DOI:
https://doi.org/10.1016/j.vetmic.2019.108507
Reference:
VETMIC 108507
To appear in:
Veterinary Microbiology
Received Date:
4 September 2019
Revised Date:
11 November 2019
Accepted Date:
12 November 2019
Please cite this article as: Fan L, Wu T, Wang Y, Hussain A, Jiang N, Gao L, Li K, Gao Y, Liu C, Cui H, Pan Q, Zhang Y, Wang X, Qi X, Novel Variants of Infectious Bursal Disease Virus Can Severely Damage the Bursa of Fabricius of Immunized Chickens, Veterinary Microbiology (2019), doi: https://doi.org/10.1016/j.vetmic.2019.108507
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Novel Variants of Infectious Bursal Disease Virus Can Severely Damage the Bursa of Fabricius of Immunized Chickens Linjin Fan a, b, Tiantian Wu a, b, Yulong Wang a, b, Altaf Hussain a, b, Nan Jiang a, b, Li Gao a, b, Kai Li a, Yulong Gao a, b, Changjun Liu a, Hongyu Cui a, Qing Pan a, Yanping Zhang a, Xiaomei Wang a, b , c, Xiaole Qi a, b * a
Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin 150069, P.R. China b OIE Reference Laboratory for Infectious Bursal Disease, Harbin 150069, P.R. China c Jiangsu Co-innovation Centre for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, P.R. China
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*To whom correspondence should be addressed: Xiaole Qi, Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 678 Haping Road, Xiangfang District, Harbin 150069, Heilongjiang Province, PR China. Tel: +86-451-51051694; Fax: +86-451-51997166; E-mail: [email protected].
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Highlights： Novel variant IBDV induced severe bursa lesions in chickens immunized with vvIBDV vaccines. The antigenic mismatch between novel variant IBDV and vvIBDV was confirmed. Key aa residues might be involved in antigenicity and virulence differences were analyzed. Unique novel variant IBDV pathogenicity allows for the prevalence of atypical IBD.
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Abstract In recent years, atypical infectious bursal disease (IBD) with severe immunosuppression has brought new threats to the poultry industry and has caused considerable economic losses. Novel variant infectious bursal disease virus (IBDV) has been identified as the etiological pathogen and for unknown reasons is widespread in poultry on many chicken farms in China that have been immunized with vaccines against very virulent IBDV (vvIBDV). Using immunoprotection experiments in specific-pathogen-free chickens, we first verified that novel variant IBDV could severely damage the bursa of Fabricius of the important immune organ of immunized chicken in the presence of antibodies induced by three types of vvIBDV vaccines, which is a primary reason for the current epidemic of atypical IBD. Monoclonal antibody reactivity patterns and cross-neutralization assays further confirmed the obvious antigenic mismatch between novel variant IBDV and vvIBDV. Sequence analysis of the genome of novel variant IBDV (SHG19 strain) was performed and the key amino acid residues that might be involved in antigenicity and 1
virulence differences of novel variant IBDV compared to vvIBDV were further analyzed. This study not only determined the primary reason for the atypical IBD epidemic, but also remind us of the urgency for developing new vaccines against novel variant IBDV.
Keywords: Novel variant infectious bursal disease virus; Immunoprotection; Antigenic variation
1. Introduction
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Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive disease in chickens (Müller et al., 2003). IBD is caused by infectious bursal disease virus (IBDV), which is an RNA virus that belongs to the genus Avibirnavirus of the family Birnaviridae. IBDV has a non-enveloped capsid structure containing a double-stranded RNA genome with two segments, A and B (Brown and Skinner, 1996; Müller et al., 2003). Segment A is approximately 3.2 kb and contains two open reading frames (ORFs) with the smaller ORF encoding the non-structural protein VP5 and the larger ORF encoding a polyprotein (PP, NH2-VP2-VP4-VP3-COOH), which is cleaved by autoproteolysis to produce viral proteins VP2, VP3, and VP4 (Luque et al., 2009). In addition to VP2 being the icosahedral capsid protein, it is also the major protective immunogen of IBDV, and the primary determinant of viral virulence and antigenic variation (Brandt et al., 2001; Fahey et al., 1989; Jackwood et al., 2008; Qi et al., 2009, 2015, 2016). Segment B is approximately 2.8 kb and contains only one ORF, which encodes viral protein VP1. As an RNA-dependent RNA polymerase (RdRp), VP1 is reported to play an important role in viral replication and genetic evolution (Escaffre et al., 2013; Gao et al., 2014; Yu et al., 2013). IBDV includes two known serotypes. Serotype I viruses are pathogenic to chickens and are further classified into four subtypes, very virulent IBDV (vvIBDV), classic IBDV, antigenic variant IBDV, and artificially attenuated IBDV strains (Müller et al., 2003). Serotype II viruses have been isolated from turkeys and are nonpathogenic to chickens. Classic IBDV was firstly identified in 1957 (Cosgrove, 1962). Subsequently, antigenic variant IBDV (Jackwood and Saif, 1987) and vvIBDV (Chettle et al., 1989) have successively emerged and provided new challenges. Over the past 30 years, vvIBDV with high fatality rate has caused considerable economic losses to the poultry industry worldwide (Jackwood, 2017; Müller et al., 2003). With the scientific advancement of vvIBDV vaccines and the improvement of biosafety measures, vvIBDV is currently being well controlled in many countries, including China. However, in recent years, atypical IBD had produced new threats to the poultry industry in China. A novel variant IBDV was originally identified by our laboratory as the pathogen of atypical IBD (Fan et al., 2019). Although no mortality is associated 2
with the novel variant IBDV, it causes severe atrophy of the bursa of the important immune organ of chickens, which results in severe immunosuppression and a loss in production performance. Importantly, many novel variant IBDVs have been isolated from immunized chickens. The objective of the current study was to determine whether novel variant IBDVs are able to escape the immunoprotection provided by vvIBDV vaccines. 2. Materials and methods 2.1. Viruses, vaccines, and monoclonal antibodies
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The novel variant IBDV representative strain SHG19 (Fan et al., 2019) was previously isolated and identified by us at the Avian Immunosuppressive Disease Laboratory, Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences (CAAS) (hereinafter referred to as “our lab”). The Chinese vvIBDV reference strains HLJ0504 (Qi et al., 2011) and Gx (Wang et al., 2004) also were previously identified by our lab. The European vvIBDV reference strain 89163 (Eterradossi et al., 1992) was kindly provided by Dr. Eterradossi of the Ploufragan-Plouzane Laboratory, French Agency for Food, Environmental and Occupational Health Safety (Anses). Three vaccines against vvIBDV, including attenuated live vaccine (Vaccine A), a subunit vaccine (Vaccine B) and combined vaccine (Vaccine C) were used in the immunoprotection experiments. Eight monoclonal antibodies (MAbs) against IBDV VP2 (1-2C-7C, 1-6H-3A, 2-3B-5D, 2-5C-6 F, 7D4, 8G, 4-5D-2E, and 3-10H-7A) were developed by our lab.
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2.2. Animals
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Specific-pathogen-free (SPF) chickens were purchased from the Experimental Animal Center of the HVRI of the CAAS (Harbin, China) and were housed in negative-pressure-filtered air isolators. All the animal experiments were approved by the HVRI of the CAAS and were performed in accordance with the animal ethics guidelines and approved protocols. 2.3. Immunoprotection experiment
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To evaluate the immunoprotective effect of the vvIBDV vaccines to novel variant IBDV, an immunoprotection experiment was performed using the three vaccines against vvIBDV. One-day-old SPF chickens were randomly divided into five groups (n=6/group). In accordance with the manufacturer's instructions, the first group was immunized with Vaccine A via the ocular and intranasal routes at ten-day-old, the second and the third groups were immunized with vaccine B and Vaccine C by intramuscular injection at ten-day-old, respectively. The fourth group was given phosphate buffer saline (PBS) as a non-immunization control. The fifth group without any immunization was used as a normal control. At 17, 24, and 27 days old, serum 3
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antibodies were detected with Infectious Bursal Disease Virus Antibody Test Kit (IDEXX). At 28-day-old, each chicken in the three immunized groups and the fourth PBS group was infected with 8 × 106 viral RNA copies of representative novel variant IBDV strain SHG19 via the ocular and intranasal route. At 5, 10 days post-infection (d p.i.), three chickens were randomly selected from each group, euthanized for necropsy, and the pathological changes were examined. The bursa weight, spleen weight, and body weight for each chicken were recorded, and the bursa:body weight index (BBIX) was calculated with the standard deviation [BBIX= (bursa:body weight ratios)/(bursa:body weight ratios in the negative group)]. The mean BBIX and standard deviations of the data obtained from three independent chicken samples were then calculated. Bursa with a BBIX less than 0.70 was considered as atrophy (Lucio and Hitchner, 1979). The spleen/body weight ratios were also determined. Part of bursa from each chicken was immediately fixed in 10% neutral buffered formalin and was stained with hematoxylin and eosin for further histopathological examination. 2.4. MAb reactivity pattern experiment
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To determine whether antigenic differences existed between novel variant IBDV (SHG19) and vvIBDV, the MAb reactivity pattern of IBDV were evaluated. The Chinese vvIBDV reference strain (HLJ0504) and European vvIBDV reference strain (89163) were selected as control. The immunofluorescence assays (IFA) in DT40 cells directed by the eight MAbs against IBDV VP2 (1-2C-7C, 1-6H-3A, 2-3B-5D, 2-5C-6F, 7D4, 8G, 4-5D-2E, and 3-10H-7A) were performed as previously described (Fan et al., 2019). 2.5. Cross-neutralization assay
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To further evaluate the antigenic differences between novel variant IBDV and vvIBDV, the antigen relations between SHG19 and the vvIBDV reference strains (Gx and HLJ0504) were analyzed by the cross-neutralization assays using the antisera against each of the three virus strains. First, the titer of 50% tissue culture infective dose (TCID50) of SHG19, Gx, and HLJ0504 strains were determined in DT40 cells by IFA using IBDV-specific MAb. Then 200 TCID50 of viruses were incubated for 1h at 37 °C with equal volumes antisera at different dilutions. The virus-sera mixture (100 μl) was then cultured with DT40 cells at 37 °C in a humidified 5% CO2 incubator. After 24 h, the infection-positive wells were detected by IFA. Cross-neutralization titers of the antisera to different viruses were calculated as described by Archetti and Horsfall (1950). The antigenic relatedness (R value) of two viruses was calculated using the formula: R2 = rl x r2, the ratio rl is determined by dividing the heterologous titer obtained with virus 2 by the homologous titer obtained with virus 1, and the ratio r2 is determined by dividing the heterologous titer obtained with virus 1 by the homologous titer obtained with virus 2. A homologous R value is defined as 1, and an R value of 1 or close to 1 indicates antigenic similarity between the two tested virus strains. The resulting R was expressed as a percentage of relatedness and was 4
interpreted as the follows: 0-0.10, a serotype difference; 0.11-0.70, a subtype difference; > 0.70, little or no difference (Chen et al., 2018; Jackwood and Saif, 1987). 2.6. Genome clone
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Total viral RNA of novel variant IBDV strain SHG19 was extracted using a PurelinkTM RNA Mini Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The first-strand complementary DNA (cDNA) was synthesized from the viral RNA using M-MLV Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA) and random primer pd(N)9. Using the cDNA as template, two fragments of the genome segment A or B were obtained by polymerase chain reaction (PCR) using the specific primers as previously described (Lu et al., 2015). The PCR products were cloned into the vector pMD18-T (Takara, China). At least three independent positive clones for each fragment were sequenced. 2.7. Sequence analysis
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2.8 Statistical analyses
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To more precisely characterize the genome of SHG19, representative IBDV strains were chosen for further analysis. The phylogenetic trees were constructed from the aligned amino acid sequences of 24 representative strains obtained from GenBank (Table 1) using the Neighbor-Joining method in MEGA 6 software (Tamura et al., 2013).
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3. Results
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A one-way ANOVA was used to evaluate the statistical significance of the differences among the different groups. And a P-value of < 0.05 was considered statistically significant.
3.1. SHG19 induced severe lesions in immunized chickens
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To detect the immunoprotective effect of vvIBDV vaccines to novel variant IBDV, the animal experiment was performed. In the animal experiment, all three immunized groups showed positive IBDV antibody with different titers at 27-day-old (Fig. 1A and Table S1). At 28-day-old, the chickens were infected with SHG19 strain of novel variant IBDV, except the chickens in the fifth group, which served as a control. Compared with the fifth group, SHG19 severely damaged the bursae at 5 d p.i. and 10 d p.i. with atrophy, yellow staining, and hard texture in the three immunized groups, which was similar to that of the non-immunized chickens in the fourth group (Fig. 1D). BBIX values also confirmed the severe atrophy of the bursae in all three immunized groups. The mean BBIX for the three groups were 0.453 ± 0.188, 0.266 ± 5
0.078, and 0.380 ± 0.043 at 5 d p.i., and 0.606 ± 0.114, 0.235 ± 0.079, and 0.293 ± 0.063 at 10 d p.i., respectively (Fig. 1B and Table S2). Based on RT-PCR and sequencing results, all challenged chickens were positive for SHG19 strain (data not shown). In addition, the spleen/body weight ratios of some of the challenged chickens were higher than those of the normal control chickens at 5 d p.i. (Fig 1C and Table S3). Furthermore, the histopathological lesions in the bursae at 5 d p.i. were also detected. Compared to the normal control, SHG19 induced decreased numbers of lymphocytes, macrophage infiltration, connective tissue hyperplasia, atrophy and destruction of follicle in the immunized groups and PBS control group (Fig. 1E). The results indicated that three different vvIBDV vaccines didn’t provide efficient protection against SHG19 challenge.
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3.2. SHG19 showed obvious antigenic difference compared with vvIBDV
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To detect the antigenic differences between novel variant IBDV and vvIBDV, the MAb reactivity pattern of IBDV were performed. In the experiment, the Chinese vvIBDV reference strain HLJ0504 and European vvIBDV reference strain 89163 each reacted with all eight MAbs (Table 2). However, only one MAb (7D4) recognized SHG19 (Table 2). The results indicated that SHG19 showed different MAb reactivity pattern from vvIBDV. The antigen relations between SHG19 and the vvIBDV reference strains were further analyzed by the cross-neutralization assays. In the tests, vvIBDV strains Gx and HLJ0504 showed antigenic similarity with an R value of 1.05 (Table 2). However, the SHG19 strain and Gx strain had an R value of 0.64, indicating antigenic subtype difference. The R value for SHG19 strain and HLJ0504 strain was 0.35, also indicating obvious antigenic difference (Table 2).
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3.3. SHG19 had distinct molecular characteristics among variant IBDVs
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To further characterize the molecular features of SHG19, the genome of SHG19 was identified and analyzed. Sequencing results showed that segment A of SHG19 strain contained 3260 nucleotides (nt), including a 5’-non-coding-region (NCR) of 84 nt, VP5 coding region of 450 nt, the PP coding region of 3039 nt and a 3’-NCR of 91 nt. Segment B was consisted of a 5’-NCR of 111 nt, VP1 coding region of 2640 nt and a 3’-NCR of 78 nt. The genome sequence of SHG19 has been submitted to GenBank with accession numbers, MN393076 for segment A and MN393077 for segment B. Phylogenetic trees based on the amino acid (aa) sequence of PP and VP1 showed that SHG19 evolutionarily belonged to variant IBDV (Fig 3). In the PP phylogenetic tree, SHG19 was located in the same branch with the American representative variant IBDVs (Variant E and 9109). For VP1, SHG19 had a greater aa identity with the American reference variant IBDVs (98.4%-98.9%) than that with vvIBDV (96.3%-97.6%). In the hypervariable region (HVR) of VP2, characteristic aa residues of the reference variant IBDVs (Variant E and 9109), including aa 213N, 222T, 242V, 249K, 256V, 253Q, 279N, 284A, 286I, 294L, 318D, 323E, and 330S, were observed 6
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in SHG19 (Fig. 4). The aa residues 222T, 249K, 286I, and 318D are considered as the typical residues of the variant IBDVs (Jackwood, 2012; Jackwood et al., 2006). However, among variant IBDVs, SHG19 also presented distinct molecular characteristics. Compared with the American representative variant IBDVs (Variant E and 9109), SHG19 had ten distinct aa residues in PP, including 73I, 77D, 79S, 187V, 221K, 252I, 299S, 451L, 922Q, and 951L. For VP5, SHG19 had only 96.4-96.8% identity with the American variants and had eight distinct aa residues, 7R, 44P, 78L, 92R, 104G, 109R, 116V and 147E. In addition, the American variants have a four-peptide (MLSL) deletion at the N-terminus of VP5 that was not present in SHG19. Also, the distinct aa residues (508K) was observed in VP1 of SHG19 (Fig. 4). Compared with vvIBDV, SHG19 showed obvious differences, not only in PP but also in VP1. In PP, except for aa residues 253, 284, 299, 330, 451, 541, 981, and 1005, the other 17 characteristic aa residues of SHG19 listed in Fig 4 differed from that of vvIBDV. These differences may be involved in the antigenic variation. In VP1, among 15 characteristic aa residues listed in Fig 4, SHG19 had 13 distinct aa residues, which may be involved in the virulence differences.
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4. Discussion
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IBD is one of the most important diseases in poultry because of the immeasurable harm that it causes. On one hand, vvIBDV causes direct economic losses due to its specific morbidity and mortality rates (Jackwood, 2017; van den Berg et al., 2000). On the other hand, the immunodeficiency that occurs in the surviving chickens increases the risk of secondary infections by other viruses, bacteria, and parasites, which compounds the damage (Berg, 2000; Zachar et al., 2016). Vaccines are an effective means of preventing and controlling vvIBDV infections. In China, almost all chicken farms use vvIBDV vaccines resulting in the disease is being largely controlled especially on large-scale intensive chicken farms with improved husbandry management. However, since 2015, atypical IBD with severe immunosuppression caused by novel variant IBDV has posed a new threaten to poultry industry (Fan et al., 2019). That was the first report of the large epidemics of variant IBDV in China. Variant IBDV is responsible for economically significant disease because it induces severe bursa damage, profound immunosuppression, and subclinical infections that are often the underlying cause of respiratory and enteric diseases in chickens, as well as being the cause of vaccination failures (Icard et al., 2008; Jackwood and Sommer-Wagner, 2005; Perozo et al., 2009). A 5-year study demonstrated that variant IBDV has had a serious influence on the Canadian economy and that it is associated with immunosuppression (Amini et al., 2015; Zachar et al., 2016). Variant IBDV is also a potential threat to antibiotic-free chicken farming and is not amenable to the current anti-IBDV vaccination strategy (Kurukulsuriya et al., 2016). A retrospective study showed that variant IBDV strains are present in backyard chickens in California, USA (Stoute et al., 2019). Our data also revealed that many novel variant IBDVs exist on chicken farms that immunized their animals with vaccines against vvIBDV in at least 7
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11 provinces in China (Fan et al., 2019), which poses another new challenge. Novel variant IBDVs might break through the immunoprotection induced by some vvIBDV vaccines. To explore this speculation, we performed an animal experiment. The judgment criterions of immunoprotection in this animal experiment is that, after 5 days post-challenge with variant IBDV, the bursa of the immunized group is normal while the bursa of the non-immunized group (PBS group) is atrophied. Results showed that the mean BBIX of each immunized group and the PBS group was less than 0.70. It is recognized that the bursa with a BBIX less than 0.70 is considered as atrophy (Lucio and Hitchner, 1979). However, the statistics sense of the differences among the BBIX values less than 0.70 is unknown. Although there are significant differences of BBIX between vaccine A group and the PBS group, chickens in vaccine A group were still identified as atrophy because the mean BBIX of vaccine A group was less than 0.70. Taking the histopathological results into consideration, there were no obvious differences in protection among three immunized groups. Taken together, we found that in the presence of IBDV antibody induced by three different vvIBDV vaccines, infection by novel variant IBDV severely damaged the bursae by inducing atrophy and lymphocytes necrocytosis. This is a primary reason of the epidemic of atypical IBD seen in immunized chickens. Antigenic variation and mismatch are primary factors involved in immune failure. It was speculated that antigenic difference existed between novel variant IBDV and vvIBDV. To verify this, the antigenic relationship between novel variant IBDV and vvIBDV were evaluated using two methods. The MAb reactivity pattern experiment showed that novel variant IBDV had different MAb reactivity patterns from not only the Chinese vvIBDV, but also the European vvIBDV. Furthermore, the antigenic mismatch of novel variant IBDV and vvIBDV were confirmed using cross-neutralization assays. The R value is a key parameter that can be used to evaluate the antigenicity between different isolates and it has practical significance in selecting strains to control avian viruses (Ladman et al., 2006). An R value below 0.7 indicates obvious antigenic difference between two virus strains (Chen et al., 2018). The novel variant IBDV strain SHG19 showed antigenic differences from vvIBDV strains Gx and HLJ0504 with R values of 0.64 and 0.35, respectively. To better understand the molecular information involved in antigenic variation and virulence of novel variant IBDV, the genome of SHG19 strain was cloned and sequenced. This was the first genome of a novel variant IBDV strain to be sequenced and analyzed. Analysis of the genome sequence further confirmed the molecular characters of SHG19 as a novel variant IBDV. It is important to note that SHG19 and vvIBDV had a relatively large genetic distance with 96.9%-97.5% aa sequence identity for PP. Among the viral proteins cleaved from PP, the capsid protein VP2 is the main determinant of virulence and antigenic variation (Brandt et al., 2001; Jackwood et al., 2008; Qi et al., 2009, 2015, 2016). VP2 is folded into three distinct domains, including the base (B), shell (S), and projection (P) domains (Birghan et al., 2000; Garriga et al., 2006; Lee et al., 2006). The HVR of VP2 (aa residues 206-350) is located in the tower-like P domain (Boot et al., 2000; Letzel et al., 2007). At the outer edge of HVR, there are four loops, the PBC (aa 204-236), PDE (aa 240-265), PFG 8
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(aa 270-293), and PHI (aa 305-337) (Coulibaly et al., 2005). Compared with vvIBDV, SHG19 had 13 characteristic aa residues in the HVR of VP2 (213N, 221K, 222T, 242V, 249K, 252I, 254N, 256V, 279N, 286I, 294L, 318D, and 323E), which might be involved in antigenic variation (Coulibaly et al., 2005; Jackwood and Sommer-Wagner, 2011; Letzel et al., 2007; Vakharia et al., 1994). It has been reported that a mutation in aa 222 changes the MAb reactivity of IBDV (Letzel et al., 2007). Even the naturally occurring mutation T222A induces immune escape of IBDV (Del-E strain) (Jackwood and Sommer-Wagner, 2011). Residue 286 (in PFG) in the hydrophilic peak B and residues 318, 321, and 323 (in PHI) in the minor peak 2 of VP2 are involved in the reactivity of both MAb 57 and MAb 67, respectively (Letzel et al., 2007; Vakharia et al., 1994). Residue 249 (in PDE) in minor peak 1 of VP2 may be the key residue in MAb B69 binding (Letzel et al., 2007; Vakharia et al., 1994). Residue 254 (in PDE) in minor peak 1 of VP2 is also related to antigenic drift of IBDV (Jackwood and Sommer-Wagner, 2011). With reverse genetics and immunology methods, the key residues involved in antigen variation and immune escape of IBDV are currently under study. It is also worthwhile to further evaluate the antigen relation in detail between SHG19-like strains and the previous known variant IBDV. In addition to antigenic related residues, aa 249 in strand PD, 253 in PDE, 256 in strand PE, and 284 in PFG of VP2 have all been identified as the main determinants of virulence (Qi et al., 2009, 2013). SHG19 and vvIBDV have residues 253Q and 284T in common, however, SHG19 differs from vvIBDV with residues of 249K and 256V. It has been reported that double mutations of Q253H and A284T reduces the mortality of vvIBDV to 0% (Qi et al., 2009). Changes of Q249R and I256V can further reduce the bursa lesions induced by vvIBDV (Qi et al., 2013). It has also been reported that both genome segments contribute to the pathogenicity of IBDV (Escaffre et al., 2013). Among the characteristic aa in VP1 that were listed in Fig 4., 87% (13/15) of the aa are the same as those found in attenuated IBDV. In VP1, residue 4 (Yu et al., 2013) and the aa triplet 145/146/147 (Gao et al., 2014) are important determinants of viral replication and pathogenicity through their effect on the RdRp activity. vvIBDV is able to kill chickens while novel variant IBDV only induces severe bursa lesions and immunosuppression, which is a mechanism that needs to be further explored. 5. Conclusions
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The current study provided for the first time evidences from both natural and laboratory data to verify that novel variant IBDV could severely damage the bursa of the important immune organ of immunized chicken. This is a primary reason for the current epidemic of atypical IBD in China. Our study further characterized the antigenic differences between the novel variant IBDV and vvIBDV. Our findings not only explained the reason for the epidemic of the atypical IBD, but also remind us of the urgency for developing new vaccines against novel variant IBDV.
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Conflict of interest statement The authors declare that they have no competing interests.
Acknowledgments
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This work was supported by the National Key Research and Development Program of China (No. 2016YFE0203200, No. 2017YFD0500704), the Heilongjiang Province Foundation for the National Key Research and Development Program of China (GX18B011), the Major Project of National Natural Science Foundation of China (No. 31430087), the Modern Agro-industry Technology Research System (No. CARS-41-G15).
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References Amini, K., Zachar, T., Popowich, S., Knezacek, T., Goodhope, B., Willson, P., Gomis, S., 2015. Association of increased rate of condemnation of broiler carcasses due to hepatic abnormalities with immunosuppressive diseases in the broiler chicken industry in Saskatchewan. Can. J. Vet. Res. 79, 261-267. Archetti, I., Horsfall, F.L., Jr., 1950. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. J Exp Med. 92, 441-462. Berg, T.P., 2000. Acute infectious bursal disease in poultry: a review. Avian Pathol. 29, 175-194. Birghan, C., Mundt, E., Gorbalenya, A.E., 2000. A non-canonical lon proteinase lacking the ATPase domain employs the ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus. EMBO J. 19, 114-123.
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Boot, H.J., ter Huurne, A.A., Hoekman, A.J., Peeters, B.P., Gielkens, A.L., 2000. Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype. J. Virol. 74, 6701-6711.
Brandt, M., Yao, K., Liu, M., Heckert, R.A., Vakharia, V.N., 2001. Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus. J. Virol. 75, 11974-11982.
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Brown, M.D., Skinner, M.A., 1996. Coding sequences of both genome segments of a European 'very virulent' infectious bursal disease virus. Virus Res. 40, 1-15.
Chen, G., He, X., Yang, L., Wei, P., 2018. Antigenicity characterization of four representative natural
re
reassortment IBDVs isolated from commercial three-yellow chickens from Southern China reveals different subtypes co-prevalent in the field. Vet. Microbiol. 219, 183-189. Vet. Rec. 125, 271-272.
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Chettle, N., Stuart, J.C., Wyeth, P.J., 1989. Outbreak of virulent infectious bursal disease in East Anglia. Cosgrove, A.S., 1962. An Apparently New Disease of Chickens: Avian Nephrosis. Avian Dis. 6, 385-389.
na
Coulibaly, F., Chevalier, C., Gutsche, I., Pous, J., Navaza, J., Bressanelli, S., Delmas, B., Rey, F.A., 2005. The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120, 761-772.
Escaffre, O., Le Nouen, C., Amelot, M., Ambroggio, X., Ogden, K.M., Guionie, O., Toquin, D., Muller,
ur
H., Islam, M.R., Eterradossi, N., 2013. Both genome segments contribute to the pathogenicity of very virulent infectious bursal disease virus. J. Virol. 87, 2767-2780. Eterradossi, N., Picault, J.P., Drouin, P., Guittet, M., L'Hospitalier, R., Bennejean, G., 1992.
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Pathogenicity and preliminary antigenic characterization of six infectious bursal disease virus strains isolated in France from acute outbreaks. Zentralbl. Veterinarmed. B. 39, 683-691.
Fahey, K.J., Erny, K., Crooks, J., 1989. A conformational immunogen on VP-2 of infectious bursal disease virus that induces virus-neutralizing antibodies that passively protect chickens. J. Gen. Virol. 70 ( Pt 6), 1473-1481. Fan, L., Wu, T., Hussain, A., Gao, Y., Zeng, X., Wang, Y., Gao, L., Li, K., Wang, Y., Liu, C., Cui, H., Pan, Q., Zhang, Y., Liu, Y., He, H., Wang, X., Qi, X., 2019. Novel variant strains of infectious bursal disease virus isolated in China. Vet. Microbiol. 230, 212-220. Gao, L., Li, K., Qi, X., Gao, H., Gao, Y., Qin, L., Wang, Y., Shen, N., Kong, X., Wang, X., 2014. Triplet 11
amino acids located at positions 145/146/147 of the RNA polymerase of very virulent infectious bursal disease virus contribute to viral virulence. J. Gen. Virol. 95, 888-897. Garriga, D., Querol-Audi, J., Abaitua, F., Saugar, I., Pous, J., Verdaguer, N., Caston, J.R., Rodriguez, J.F., 2006. The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid. J. Virol. 80, 6895-6905. Icard, A.H., Sellers, H.S., Mundt, E., 2008. Detection of infectious bursal disease virus isolates with unknown antigenic properties by reverse genetics. Avian Dis. 52, 590-598. Jackwood, D.H., Saif, Y.M., 1987. Antigenic diversity of infectious bursal disease viruses. Avian Dis. 31, 766-770. Jackwood, D.J., 2012. Molecular epidemiologic evidence of homologous recombination in infectious bursal disease viruses. Avian Dis. 56, 574-577. Jackwood, D.J., 2017. Advances in vaccine research against economically important viral diseases of food animals: Infectious bursal disease virus. Vet. Microbiol. 206, 121-125.
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Jackwood, D.J., Cookson, K.C., Sommer-Wagner, S.E., Le Galludec, H., de Wit, J.J., 2006. Molecular characteristics of infectious bursal disease viruses from asymptomatic broiler flocks in Europe. Avian Dis. 50, 532-536.
Jackwood, D.J., Sommer-Wagner, S.E., 2005. Molecular epidemiology of infectious bursal disease
viruses: distribution and genetic analysis of newly emerging viruses in the United States. Avian
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Dis. 49, 220-226.
Jackwood, D.J., Sommer-Wagner, S.E., 2011. Amino acids contributing to antigenic drift in the infectious bursal disease Birnavirus (IBDV). Virology 409, 33-37.
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Jackwood, D.J., Sreedevi, B., LeFever, L.J., Sommer-Wagner, S.E., 2008. Studies on naturally occurring infectious bursal disease viruses suggest that a single amino acid substitution at position 253 in VP2 increases pathogenicity. Virology 377, 110-116.
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Kurukulsuriya, S., Ahmed, K.A., Ojkic, D., Gunawardana, T., Gupta, A., Goonewardene, K., Karunaratne, R., Popowich, S., Willson, P., Tikoo, S.K., Gomis, S., 2016. Circulating strains of variant infectious bursal disease virus may pose a challenge for antibiotic-free chicken farming in Canada. Res.Vet. Sci. 108, 54-59.
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Ladman, B.S., Loupos, A.B., Gelb, J., Jr., 2006. Infectious bronchitis virus S1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization. Avian Pathol. 35, 127-133.
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Lee, C.C., Ko, T.P., Chou, C.C., Yoshimura, M., Doong, S.R., Wang, M.Y., Wang, A.H., 2006. Crystal structure of infectious bursal disease virus VP2 subviral particle at 2.6A resolution: implications in virion assembly and immunogenicity. J. Struct. Biol. 155, 74-86.
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Letzel, T., Coulibaly, F., Rey, F.A., Delmas, B., Jagt, E., van Loon, A.A., Mundt, E., 2007. Molecular and structural bases for the antigenicity of VP2 of infectious bursal disease virus. J. Virol. 81, 12827-12835.
Lu, Z., Zhang, L., Wang, N., Chen, Y., Gao, L., Wang, Y., Gao, H., Gao, Y., Li, K., Qi, X., Wang, X., 2015. Naturally occurring reassortant infectious bursal disease virus in northern China. Virus Res. 203, 92-95. Lucio, B., Hitchner, S.B., 1979. Response of susceptible versus immune chicks to killed, live-modified, and wild infectious bursal disease virus vaccines. Avian Dis. 23, 1037-1050. Luque, D., Rivas, G., Alfonso, C., Carrascosa, J.L., Rodriguez, J.F., Caston, J.R., 2009. Infectious bursal disease virus is an icosahedral polyploid dsRNA virus. Proc. Natl. Acad. Sci. U S A. 106, 12
2148-2152. Müller, H., Islam, M.R., Raue, R., 2003. Research on infectious bursal disease—the past, the present and the future. Vet. Microbiol. 97, 153-165. Perozo, F., Villegas, A.P., Fernandez, R., Cruz, J., Pritchard, N., 2009. Efficacy of single dose recombinant herpesvirus of turkey infectious bursal disease virus (IBDV) vaccination against a variant IBDV strain. Avian Dis. 53, 624-628. Qi, X., Gao, H., Gao, Y., Qin, L., Wang, Y., Gao, L., Wang, X., 2009. Naturally occurring mutations at residues 253 and 284 in VP2 contribute to the cell tropism and virulence of very virulent infectious bursal disease virus. Antiviral Res. 84, 225-233. Qi, X., Gao, L., Qin, L., Deng, X., Wu, G., Zhang, L., Fei, Y., Ren, X., Gao, Y., Gao, H., Wang, Y., Wang, X., 2011. Genomic Sequencing and Molecular Characteristics of A Very Virulent Strain of Infectious Bursal Disease Virus Isolated in China. Agric. Sci. Technol. 16, 1565-1569. Qi, X., Gao, X., Lu, Z., Zhang, L., Wang, Y., Gao, L., Gao, Y., Li, K., Gao, H., Liu, C., Cui, H., Zhang,
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Y., Wang, X., 2016. A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus. Sci. China Life Sci. 59, 717-723.
Qi, X., Qin, L., Gao, Y., Gao, H., Li, Y., Li, G., Lu, Z., Wang, N., Chen, Y., Zhang, L., Li, K., Wang, Y., Wang, X., 2015. Genetic Analysis of the VP2 Hypervariable Region of Thirty-six Infectious
Bursal Disease Virus Isolates in China during 2009-2012. Agric. Sci. Technol. 12, 1950-1953.
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Qi, X., Zhang, L., Chen, Y., Gao, L., Wu, G., Qin, L., Wang, Y., Ren, X., Gao, Y., Gao, H., Wang, X.,
2013. Mutations of residues 249 and 256 in VP2 are involved in the replication and virulence of infectious Bursal disease virus. PloS one 8, e70982.
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Stoute, S.T., Jackwood, D.J., Crossley, B.M., Michel, L.O., Blakey, J.R., 2019. Molecular epidemiology of endemic and very virulent infectious bursal disease virus genogroups in backyard chickens in California, 2009-2017. J. Vet. Diagn. Invest. 31, 371-377.
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Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729. Vakharia, V.N., He, J., Ahamed, B., Snyder, D.B., 1994. Molecular basis of antigenic variation in infectious bursal disease virus. Virus Res. 31, 265-273.
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van den Berg, T.P., Eterradossi, N., Toquin, D., Meulemans, G., 2000. Infectious bursal disease (Gumboro disease). Rev. Sci. Tech. 19, 509-543. Wang, X.M., Zeng, X.W., Gao, H.L., Fu, C.Y., Wei, P., 2004. Changes in VP2 gene during the
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attenuation of very virulent infectious bursal disease virus strain Gx isolated in China. Avian Dis. 48, 77-83.
Yu, F., Ren, X., Wang, Y., Qi, X., Song, J., Gao, Y., Qin, L., Gao, H., Wang, X., 2013. A single amino
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acid V4I substitution in VP1 attenuates virulence of very virulent infectious bursal disease virus (vvIBDV) in SPF chickens and increases replication in CEF cells. Virology 440, 204-209. Zachar, T., Popowich, S., Goodhope, B., Knezacek, T., Ojkic, D., Willson, P., Ahmed, K.A., Gomis, S., 2016. A 5-year study of the incidence and economic impact of variant infectious bursal disease viruses on broiler production in Saskatchewan, Canada. Can. J. Vet. Res. 80, 255-261.
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Figure legends
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Fig. 1. Immunoprotective evaluation of vvIBDV vaccines against novel variant IBDV strain SHG19 using specific-pathogen-free (SPF) Chickens. A. IBDV-specific antibody induced by three types of vvIBDV vaccines prior to challenge. B. The bursa:body weight index (BBIX) at 5 and 10 days post-infection (d p.i.). C. The spleen/body weight ratio. D. The bursae of different groups. E. The histopathological appearance of the bursal sections (hematoxylin and eosin staining). The mean titers and standard deviations (error bars) from six (A) or three (B, C) independent samples are shown. ∗ and ∗∗ represent P < 0.05 and P < 0.01 compared with PBS (B) group or control group (C).
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Fig. 2. Phylogenetic tree analysis of amino acid sequences of the viral polyprotein (A) and VP1 (B). The trees were generated by the neighbor-joining method using MEGA6 with 1000 replications. The novel variant IBDV strain SHG19 is highlighted with the solid circular.
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Fig. 3. Characteristic amino acid substitutions in VP5, polyprotein, and VP1 of variant IBDV and vvIBDV. Var, variant; VV, very virulent; AT, attenuated strain. Asterisks indicate residues identical to the sequence of novel variant IBDV strain SHG19.
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Table 1 Reference IBDV strains. Phenotype
OKYM HK46 D6948 KS UK661 89163 Gx HLJ0504 HuB-1 BD399 Variant E 9109
Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Variant Variant
GenBank accession no.
IBDV Strains
Segment A
Segment B
D49706 AF092943 AF240686 DQ927042 NC-004178 HG974563 AY444873 GQ451330 KF569805 AF362776 AF133904 AY462027
D49707 AF092944 AF240687 DQ927043 NC-004179 HG974564 AY705393 GQ451331 GQ449693 AF362770 AF133905 AY459321
Phenotype
IM F52/70 CU-1 Gt CT P2 NB CEF-94 HZ2 D78 JD1 OH
Classic Classic Attenuated Attenuated Attenuated Attenuated Attenuated Attenuated Attenuated Attenuated
GenBank accession no. Segment A
Segment B
AY029166 HG974565 X16107 DQ403248 AJ310185 X84034 AY319768 AF133904 AF321054 AF499929 AF321055 U30818
AY029165 HG974566 AF362775 DQ403249 AJ310186 X84035 AY654284 AF133905 AF493979 AF499930 AY103464 U30819
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IBDV Strains
Attenuated
Serotype II
1-6H-3A
2-3B-5D
89163
+ +
+ +
+ +
SHG19
—
—
—
+ + —
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HLJ0504
2-5C-6F
7D4
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1-2C-7C
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Table 2 Comparison of MAb reactivity pattern of novel variant IBDV strain SHG19 and vvIBDV.
+ + +
8G
4-5D-2E
3-10H-7A
+ +
+ +
+ +
—
—
—
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Note: “+” and “-” indicate the positive and negative reaction signal of the immunofluorescence assays.
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Table 3 R value of cross-neutralization assays. SHG19 Gx HLJ0504 SHG19 1.00 Gx 0.64 1.00 HLJ0504 0.35 1.05 1.00
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PII:
S0378-1135(19)31053-3
DOI:
https://doi.org/10.1016/j.vetmic.2019.108507
Reference:
VETMIC 108507
To appear in:
Veterinary Microbiology
Received Date:
4 September 2019
Revised Date:
11 November 2019
Accepted Date:
12 November 2019
Please cite this article as: Fan L, Wu T, Wang Y, Hussain A, Jiang N, Gao L, Li K, Gao Y, Liu C, Cui H, Pan Q, Zhang Y, Wang X, Qi X, Novel Variants of Infectious Bursal Disease Virus Can Severely Damage the Bursa of Fabricius of Immunized Chickens, Veterinary Microbiology (2019), doi: https://doi.org/10.1016/j.vetmic.2019.108507
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Novel Variants of Infectious Bursal Disease Virus Can Severely Damage the Bursa of Fabricius of Immunized Chickens Linjin Fan a, b, Tiantian Wu a, b, Yulong Wang a, b, Altaf Hussain a, b, Nan Jiang a, b, Li Gao a, b, Kai Li a, Yulong Gao a, b, Changjun Liu a, Hongyu Cui a, Qing Pan a, Yanping Zhang a, Xiaomei Wang a, b , c, Xiaole Qi a, b * a
Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin 150069, P.R. China b OIE Reference Laboratory for Infectious Bursal Disease, Harbin 150069, P.R. China c Jiangsu Co-innovation Centre for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, P.R. China
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*To whom correspondence should be addressed: Xiaole Qi, Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 678 Haping Road, Xiangfang District, Harbin 150069, Heilongjiang Province, PR China. Tel: +86-451-51051694; Fax: +86-451-51997166; E-mail: [email protected].
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Highlights： Novel variant IBDV induced severe bursa lesions in chickens immunized with vvIBDV vaccines. The antigenic mismatch between novel variant IBDV and vvIBDV was confirmed. Key aa residues might be involved in antigenicity and virulence differences were analyzed. Unique novel variant IBDV pathogenicity allows for the prevalence of atypical IBD.
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Abstract In recent years, atypical infectious bursal disease (IBD) with severe immunosuppression has brought new threats to the poultry industry and has caused considerable economic losses. Novel variant infectious bursal disease virus (IBDV) has been identified as the etiological pathogen and for unknown reasons is widespread in poultry on many chicken farms in China that have been immunized with vaccines against very virulent IBDV (vvIBDV). Using immunoprotection experiments in specific-pathogen-free chickens, we first verified that novel variant IBDV could severely damage the bursa of Fabricius of the important immune organ of immunized chicken in the presence of antibodies induced by three types of vvIBDV vaccines, which is a primary reason for the current epidemic of atypical IBD. Monoclonal antibody reactivity patterns and cross-neutralization assays further confirmed the obvious antigenic mismatch between novel variant IBDV and vvIBDV. Sequence analysis of the genome of novel variant IBDV (SHG19 strain) was performed and the key amino acid residues that might be involved in antigenicity and 1
virulence differences of novel variant IBDV compared to vvIBDV were further analyzed. This study not only determined the primary reason for the atypical IBD epidemic, but also remind us of the urgency for developing new vaccines against novel variant IBDV.
Keywords: Novel variant infectious bursal disease virus; Immunoprotection; Antigenic variation
1. Introduction
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Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive disease in chickens (Müller et al., 2003). IBD is caused by infectious bursal disease virus (IBDV), which is an RNA virus that belongs to the genus Avibirnavirus of the family Birnaviridae. IBDV has a non-enveloped capsid structure containing a double-stranded RNA genome with two segments, A and B (Brown and Skinner, 1996; Müller et al., 2003). Segment A is approximately 3.2 kb and contains two open reading frames (ORFs) with the smaller ORF encoding the non-structural protein VP5 and the larger ORF encoding a polyprotein (PP, NH2-VP2-VP4-VP3-COOH), which is cleaved by autoproteolysis to produce viral proteins VP2, VP3, and VP4 (Luque et al., 2009). In addition to VP2 being the icosahedral capsid protein, it is also the major protective immunogen of IBDV, and the primary determinant of viral virulence and antigenic variation (Brandt et al., 2001; Fahey et al., 1989; Jackwood et al., 2008; Qi et al., 2009, 2015, 2016). Segment B is approximately 2.8 kb and contains only one ORF, which encodes viral protein VP1. As an RNA-dependent RNA polymerase (RdRp), VP1 is reported to play an important role in viral replication and genetic evolution (Escaffre et al., 2013; Gao et al., 2014; Yu et al., 2013). IBDV includes two known serotypes. Serotype I viruses are pathogenic to chickens and are further classified into four subtypes, very virulent IBDV (vvIBDV), classic IBDV, antigenic variant IBDV, and artificially attenuated IBDV strains (Müller et al., 2003). Serotype II viruses have been isolated from turkeys and are nonpathogenic to chickens. Classic IBDV was firstly identified in 1957 (Cosgrove, 1962). Subsequently, antigenic variant IBDV (Jackwood and Saif, 1987) and vvIBDV (Chettle et al., 1989) have successively emerged and provided new challenges. Over the past 30 years, vvIBDV with high fatality rate has caused considerable economic losses to the poultry industry worldwide (Jackwood, 2017; Müller et al., 2003). With the scientific advancement of vvIBDV vaccines and the improvement of biosafety measures, vvIBDV is currently being well controlled in many countries, including China. However, in recent years, atypical IBD had produced new threats to the poultry industry in China. A novel variant IBDV was originally identified by our laboratory as the pathogen of atypical IBD (Fan et al., 2019). Although no mortality is associated 2
with the novel variant IBDV, it causes severe atrophy of the bursa of the important immune organ of chickens, which results in severe immunosuppression and a loss in production performance. Importantly, many novel variant IBDVs have been isolated from immunized chickens. The objective of the current study was to determine whether novel variant IBDVs are able to escape the immunoprotection provided by vvIBDV vaccines. 2. Materials and methods 2.1. Viruses, vaccines, and monoclonal antibodies
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The novel variant IBDV representative strain SHG19 (Fan et al., 2019) was previously isolated and identified by us at the Avian Immunosuppressive Disease Laboratory, Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences (CAAS) (hereinafter referred to as “our lab”). The Chinese vvIBDV reference strains HLJ0504 (Qi et al., 2011) and Gx (Wang et al., 2004) also were previously identified by our lab. The European vvIBDV reference strain 89163 (Eterradossi et al., 1992) was kindly provided by Dr. Eterradossi of the Ploufragan-Plouzane Laboratory, French Agency for Food, Environmental and Occupational Health Safety (Anses). Three vaccines against vvIBDV, including attenuated live vaccine (Vaccine A), a subunit vaccine (Vaccine B) and combined vaccine (Vaccine C) were used in the immunoprotection experiments. Eight monoclonal antibodies (MAbs) against IBDV VP2 (1-2C-7C, 1-6H-3A, 2-3B-5D, 2-5C-6 F, 7D4, 8G, 4-5D-2E, and 3-10H-7A) were developed by our lab.
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2.2. Animals
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Specific-pathogen-free (SPF) chickens were purchased from the Experimental Animal Center of the HVRI of the CAAS (Harbin, China) and were housed in negative-pressure-filtered air isolators. All the animal experiments were approved by the HVRI of the CAAS and were performed in accordance with the animal ethics guidelines and approved protocols. 2.3. Immunoprotection experiment
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To evaluate the immunoprotective effect of the vvIBDV vaccines to novel variant IBDV, an immunoprotection experiment was performed using the three vaccines against vvIBDV. One-day-old SPF chickens were randomly divided into five groups (n=6/group). In accordance with the manufacturer's instructions, the first group was immunized with Vaccine A via the ocular and intranasal routes at ten-day-old, the second and the third groups were immunized with vaccine B and Vaccine C by intramuscular injection at ten-day-old, respectively. The fourth group was given phosphate buffer saline (PBS) as a non-immunization control. The fifth group without any immunization was used as a normal control. At 17, 24, and 27 days old, serum 3
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antibodies were detected with Infectious Bursal Disease Virus Antibody Test Kit (IDEXX). At 28-day-old, each chicken in the three immunized groups and the fourth PBS group was infected with 8 × 106 viral RNA copies of representative novel variant IBDV strain SHG19 via the ocular and intranasal route. At 5, 10 days post-infection (d p.i.), three chickens were randomly selected from each group, euthanized for necropsy, and the pathological changes were examined. The bursa weight, spleen weight, and body weight for each chicken were recorded, and the bursa:body weight index (BBIX) was calculated with the standard deviation [BBIX= (bursa:body weight ratios)/(bursa:body weight ratios in the negative group)]. The mean BBIX and standard deviations of the data obtained from three independent chicken samples were then calculated. Bursa with a BBIX less than 0.70 was considered as atrophy (Lucio and Hitchner, 1979). The spleen/body weight ratios were also determined. Part of bursa from each chicken was immediately fixed in 10% neutral buffered formalin and was stained with hematoxylin and eosin for further histopathological examination. 2.4. MAb reactivity pattern experiment
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To determine whether antigenic differences existed between novel variant IBDV (SHG19) and vvIBDV, the MAb reactivity pattern of IBDV were evaluated. The Chinese vvIBDV reference strain (HLJ0504) and European vvIBDV reference strain (89163) were selected as control. The immunofluorescence assays (IFA) in DT40 cells directed by the eight MAbs against IBDV VP2 (1-2C-7C, 1-6H-3A, 2-3B-5D, 2-5C-6F, 7D4, 8G, 4-5D-2E, and 3-10H-7A) were performed as previously described (Fan et al., 2019). 2.5. Cross-neutralization assay
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To further evaluate the antigenic differences between novel variant IBDV and vvIBDV, the antigen relations between SHG19 and the vvIBDV reference strains (Gx and HLJ0504) were analyzed by the cross-neutralization assays using the antisera against each of the three virus strains. First, the titer of 50% tissue culture infective dose (TCID50) of SHG19, Gx, and HLJ0504 strains were determined in DT40 cells by IFA using IBDV-specific MAb. Then 200 TCID50 of viruses were incubated for 1h at 37 °C with equal volumes antisera at different dilutions. The virus-sera mixture (100 μl) was then cultured with DT40 cells at 37 °C in a humidified 5% CO2 incubator. After 24 h, the infection-positive wells were detected by IFA. Cross-neutralization titers of the antisera to different viruses were calculated as described by Archetti and Horsfall (1950). The antigenic relatedness (R value) of two viruses was calculated using the formula: R2 = rl x r2, the ratio rl is determined by dividing the heterologous titer obtained with virus 2 by the homologous titer obtained with virus 1, and the ratio r2 is determined by dividing the heterologous titer obtained with virus 1 by the homologous titer obtained with virus 2. A homologous R value is defined as 1, and an R value of 1 or close to 1 indicates antigenic similarity between the two tested virus strains. The resulting R was expressed as a percentage of relatedness and was 4
interpreted as the follows: 0-0.10, a serotype difference; 0.11-0.70, a subtype difference; > 0.70, little or no difference (Chen et al., 2018; Jackwood and Saif, 1987). 2.6. Genome clone
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Total viral RNA of novel variant IBDV strain SHG19 was extracted using a PurelinkTM RNA Mini Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The first-strand complementary DNA (cDNA) was synthesized from the viral RNA using M-MLV Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA) and random primer pd(N)9. Using the cDNA as template, two fragments of the genome segment A or B were obtained by polymerase chain reaction (PCR) using the specific primers as previously described (Lu et al., 2015). The PCR products were cloned into the vector pMD18-T (Takara, China). At least three independent positive clones for each fragment were sequenced. 2.7. Sequence analysis
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To more precisely characterize the genome of SHG19, representative IBDV strains were chosen for further analysis. The phylogenetic trees were constructed from the aligned amino acid sequences of 24 representative strains obtained from GenBank (Table 1) using the Neighbor-Joining method in MEGA 6 software (Tamura et al., 2013).
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3. Results
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A one-way ANOVA was used to evaluate the statistical significance of the differences among the different groups. And a P-value of < 0.05 was considered statistically significant.
3.1. SHG19 induced severe lesions in immunized chickens
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To detect the immunoprotective effect of vvIBDV vaccines to novel variant IBDV, the animal experiment was performed. In the animal experiment, all three immunized groups showed positive IBDV antibody with different titers at 27-day-old (Fig. 1A and Table S1). At 28-day-old, the chickens were infected with SHG19 strain of novel variant IBDV, except the chickens in the fifth group, which served as a control. Compared with the fifth group, SHG19 severely damaged the bursae at 5 d p.i. and 10 d p.i. with atrophy, yellow staining, and hard texture in the three immunized groups, which was similar to that of the non-immunized chickens in the fourth group (Fig. 1D). BBIX values also confirmed the severe atrophy of the bursae in all three immunized groups. The mean BBIX for the three groups were 0.453 ± 0.188, 0.266 ± 5
0.078, and 0.380 ± 0.043 at 5 d p.i., and 0.606 ± 0.114, 0.235 ± 0.079, and 0.293 ± 0.063 at 10 d p.i., respectively (Fig. 1B and Table S2). Based on RT-PCR and sequencing results, all challenged chickens were positive for SHG19 strain (data not shown). In addition, the spleen/body weight ratios of some of the challenged chickens were higher than those of the normal control chickens at 5 d p.i. (Fig 1C and Table S3). Furthermore, the histopathological lesions in the bursae at 5 d p.i. were also detected. Compared to the normal control, SHG19 induced decreased numbers of lymphocytes, macrophage infiltration, connective tissue hyperplasia, atrophy and destruction of follicle in the immunized groups and PBS control group (Fig. 1E). The results indicated that three different vvIBDV vaccines didn’t provide efficient protection against SHG19 challenge.
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3.2. SHG19 showed obvious antigenic difference compared with vvIBDV
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To detect the antigenic differences between novel variant IBDV and vvIBDV, the MAb reactivity pattern of IBDV were performed. In the experiment, the Chinese vvIBDV reference strain HLJ0504 and European vvIBDV reference strain 89163 each reacted with all eight MAbs (Table 2). However, only one MAb (7D4) recognized SHG19 (Table 2). The results indicated that SHG19 showed different MAb reactivity pattern from vvIBDV. The antigen relations between SHG19 and the vvIBDV reference strains were further analyzed by the cross-neutralization assays. In the tests, vvIBDV strains Gx and HLJ0504 showed antigenic similarity with an R value of 1.05 (Table 2). However, the SHG19 strain and Gx strain had an R value of 0.64, indicating antigenic subtype difference. The R value for SHG19 strain and HLJ0504 strain was 0.35, also indicating obvious antigenic difference (Table 2).
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3.3. SHG19 had distinct molecular characteristics among variant IBDVs
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To further characterize the molecular features of SHG19, the genome of SHG19 was identified and analyzed. Sequencing results showed that segment A of SHG19 strain contained 3260 nucleotides (nt), including a 5’-non-coding-region (NCR) of 84 nt, VP5 coding region of 450 nt, the PP coding region of 3039 nt and a 3’-NCR of 91 nt. Segment B was consisted of a 5’-NCR of 111 nt, VP1 coding region of 2640 nt and a 3’-NCR of 78 nt. The genome sequence of SHG19 has been submitted to GenBank with accession numbers, MN393076 for segment A and MN393077 for segment B. Phylogenetic trees based on the amino acid (aa) sequence of PP and VP1 showed that SHG19 evolutionarily belonged to variant IBDV (Fig 3). In the PP phylogenetic tree, SHG19 was located in the same branch with the American representative variant IBDVs (Variant E and 9109). For VP1, SHG19 had a greater aa identity with the American reference variant IBDVs (98.4%-98.9%) than that with vvIBDV (96.3%-97.6%). In the hypervariable region (HVR) of VP2, characteristic aa residues of the reference variant IBDVs (Variant E and 9109), including aa 213N, 222T, 242V, 249K, 256V, 253Q, 279N, 284A, 286I, 294L, 318D, 323E, and 330S, were observed 6
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in SHG19 (Fig. 4). The aa residues 222T, 249K, 286I, and 318D are considered as the typical residues of the variant IBDVs (Jackwood, 2012; Jackwood et al., 2006). However, among variant IBDVs, SHG19 also presented distinct molecular characteristics. Compared with the American representative variant IBDVs (Variant E and 9109), SHG19 had ten distinct aa residues in PP, including 73I, 77D, 79S, 187V, 221K, 252I, 299S, 451L, 922Q, and 951L. For VP5, SHG19 had only 96.4-96.8% identity with the American variants and had eight distinct aa residues, 7R, 44P, 78L, 92R, 104G, 109R, 116V and 147E. In addition, the American variants have a four-peptide (MLSL) deletion at the N-terminus of VP5 that was not present in SHG19. Also, the distinct aa residues (508K) was observed in VP1 of SHG19 (Fig. 4). Compared with vvIBDV, SHG19 showed obvious differences, not only in PP but also in VP1. In PP, except for aa residues 253, 284, 299, 330, 451, 541, 981, and 1005, the other 17 characteristic aa residues of SHG19 listed in Fig 4 differed from that of vvIBDV. These differences may be involved in the antigenic variation. In VP1, among 15 characteristic aa residues listed in Fig 4, SHG19 had 13 distinct aa residues, which may be involved in the virulence differences.
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4. Discussion
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IBD is one of the most important diseases in poultry because of the immeasurable harm that it causes. On one hand, vvIBDV causes direct economic losses due to its specific morbidity and mortality rates (Jackwood, 2017; van den Berg et al., 2000). On the other hand, the immunodeficiency that occurs in the surviving chickens increases the risk of secondary infections by other viruses, bacteria, and parasites, which compounds the damage (Berg, 2000; Zachar et al., 2016). Vaccines are an effective means of preventing and controlling vvIBDV infections. In China, almost all chicken farms use vvIBDV vaccines resulting in the disease is being largely controlled especially on large-scale intensive chicken farms with improved husbandry management. However, since 2015, atypical IBD with severe immunosuppression caused by novel variant IBDV has posed a new threaten to poultry industry (Fan et al., 2019). That was the first report of the large epidemics of variant IBDV in China. Variant IBDV is responsible for economically significant disease because it induces severe bursa damage, profound immunosuppression, and subclinical infections that are often the underlying cause of respiratory and enteric diseases in chickens, as well as being the cause of vaccination failures (Icard et al., 2008; Jackwood and Sommer-Wagner, 2005; Perozo et al., 2009). A 5-year study demonstrated that variant IBDV has had a serious influence on the Canadian economy and that it is associated with immunosuppression (Amini et al., 2015; Zachar et al., 2016). Variant IBDV is also a potential threat to antibiotic-free chicken farming and is not amenable to the current anti-IBDV vaccination strategy (Kurukulsuriya et al., 2016). A retrospective study showed that variant IBDV strains are present in backyard chickens in California, USA (Stoute et al., 2019). Our data also revealed that many novel variant IBDVs exist on chicken farms that immunized their animals with vaccines against vvIBDV in at least 7
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11 provinces in China (Fan et al., 2019), which poses another new challenge. Novel variant IBDVs might break through the immunoprotection induced by some vvIBDV vaccines. To explore this speculation, we performed an animal experiment. The judgment criterions of immunoprotection in this animal experiment is that, after 5 days post-challenge with variant IBDV, the bursa of the immunized group is normal while the bursa of the non-immunized group (PBS group) is atrophied. Results showed that the mean BBIX of each immunized group and the PBS group was less than 0.70. It is recognized that the bursa with a BBIX less than 0.70 is considered as atrophy (Lucio and Hitchner, 1979). However, the statistics sense of the differences among the BBIX values less than 0.70 is unknown. Although there are significant differences of BBIX between vaccine A group and the PBS group, chickens in vaccine A group were still identified as atrophy because the mean BBIX of vaccine A group was less than 0.70. Taking the histopathological results into consideration, there were no obvious differences in protection among three immunized groups. Taken together, we found that in the presence of IBDV antibody induced by three different vvIBDV vaccines, infection by novel variant IBDV severely damaged the bursae by inducing atrophy and lymphocytes necrocytosis. This is a primary reason of the epidemic of atypical IBD seen in immunized chickens. Antigenic variation and mismatch are primary factors involved in immune failure. It was speculated that antigenic difference existed between novel variant IBDV and vvIBDV. To verify this, the antigenic relationship between novel variant IBDV and vvIBDV were evaluated using two methods. The MAb reactivity pattern experiment showed that novel variant IBDV had different MAb reactivity patterns from not only the Chinese vvIBDV, but also the European vvIBDV. Furthermore, the antigenic mismatch of novel variant IBDV and vvIBDV were confirmed using cross-neutralization assays. The R value is a key parameter that can be used to evaluate the antigenicity between different isolates and it has practical significance in selecting strains to control avian viruses (Ladman et al., 2006). An R value below 0.7 indicates obvious antigenic difference between two virus strains (Chen et al., 2018). The novel variant IBDV strain SHG19 showed antigenic differences from vvIBDV strains Gx and HLJ0504 with R values of 0.64 and 0.35, respectively. To better understand the molecular information involved in antigenic variation and virulence of novel variant IBDV, the genome of SHG19 strain was cloned and sequenced. This was the first genome of a novel variant IBDV strain to be sequenced and analyzed. Analysis of the genome sequence further confirmed the molecular characters of SHG19 as a novel variant IBDV. It is important to note that SHG19 and vvIBDV had a relatively large genetic distance with 96.9%-97.5% aa sequence identity for PP. Among the viral proteins cleaved from PP, the capsid protein VP2 is the main determinant of virulence and antigenic variation (Brandt et al., 2001; Jackwood et al., 2008; Qi et al., 2009, 2015, 2016). VP2 is folded into three distinct domains, including the base (B), shell (S), and projection (P) domains (Birghan et al., 2000; Garriga et al., 2006; Lee et al., 2006). The HVR of VP2 (aa residues 206-350) is located in the tower-like P domain (Boot et al., 2000; Letzel et al., 2007). At the outer edge of HVR, there are four loops, the PBC (aa 204-236), PDE (aa 240-265), PFG 8
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(aa 270-293), and PHI (aa 305-337) (Coulibaly et al., 2005). Compared with vvIBDV, SHG19 had 13 characteristic aa residues in the HVR of VP2 (213N, 221K, 222T, 242V, 249K, 252I, 254N, 256V, 279N, 286I, 294L, 318D, and 323E), which might be involved in antigenic variation (Coulibaly et al., 2005; Jackwood and Sommer-Wagner, 2011; Letzel et al., 2007; Vakharia et al., 1994). It has been reported that a mutation in aa 222 changes the MAb reactivity of IBDV (Letzel et al., 2007). Even the naturally occurring mutation T222A induces immune escape of IBDV (Del-E strain) (Jackwood and Sommer-Wagner, 2011). Residue 286 (in PFG) in the hydrophilic peak B and residues 318, 321, and 323 (in PHI) in the minor peak 2 of VP2 are involved in the reactivity of both MAb 57 and MAb 67, respectively (Letzel et al., 2007; Vakharia et al., 1994). Residue 249 (in PDE) in minor peak 1 of VP2 may be the key residue in MAb B69 binding (Letzel et al., 2007; Vakharia et al., 1994). Residue 254 (in PDE) in minor peak 1 of VP2 is also related to antigenic drift of IBDV (Jackwood and Sommer-Wagner, 2011). With reverse genetics and immunology methods, the key residues involved in antigen variation and immune escape of IBDV are currently under study. It is also worthwhile to further evaluate the antigen relation in detail between SHG19-like strains and the previous known variant IBDV. In addition to antigenic related residues, aa 249 in strand PD, 253 in PDE, 256 in strand PE, and 284 in PFG of VP2 have all been identified as the main determinants of virulence (Qi et al., 2009, 2013). SHG19 and vvIBDV have residues 253Q and 284T in common, however, SHG19 differs from vvIBDV with residues of 249K and 256V. It has been reported that double mutations of Q253H and A284T reduces the mortality of vvIBDV to 0% (Qi et al., 2009). Changes of Q249R and I256V can further reduce the bursa lesions induced by vvIBDV (Qi et al., 2013). It has also been reported that both genome segments contribute to the pathogenicity of IBDV (Escaffre et al., 2013). Among the characteristic aa in VP1 that were listed in Fig 4., 87% (13/15) of the aa are the same as those found in attenuated IBDV. In VP1, residue 4 (Yu et al., 2013) and the aa triplet 145/146/147 (Gao et al., 2014) are important determinants of viral replication and pathogenicity through their effect on the RdRp activity. vvIBDV is able to kill chickens while novel variant IBDV only induces severe bursa lesions and immunosuppression, which is a mechanism that needs to be further explored. 5. Conclusions
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The current study provided for the first time evidences from both natural and laboratory data to verify that novel variant IBDV could severely damage the bursa of the important immune organ of immunized chicken. This is a primary reason for the current epidemic of atypical IBD in China. Our study further characterized the antigenic differences between the novel variant IBDV and vvIBDV. Our findings not only explained the reason for the epidemic of the atypical IBD, but also remind us of the urgency for developing new vaccines against novel variant IBDV.
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Conflict of interest statement The authors declare that they have no competing interests.
Acknowledgments
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This work was supported by the National Key Research and Development Program of China (No. 2016YFE0203200, No. 2017YFD0500704), the Heilongjiang Province Foundation for the National Key Research and Development Program of China (GX18B011), the Major Project of National Natural Science Foundation of China (No. 31430087), the Modern Agro-industry Technology Research System (No. CARS-41-G15).
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References Amini, K., Zachar, T., Popowich, S., Knezacek, T., Goodhope, B., Willson, P., Gomis, S., 2015. Association of increased rate of condemnation of broiler carcasses due to hepatic abnormalities with immunosuppressive diseases in the broiler chicken industry in Saskatchewan. Can. J. Vet. Res. 79, 261-267. Archetti, I., Horsfall, F.L., Jr., 1950. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. J Exp Med. 92, 441-462. Berg, T.P., 2000. Acute infectious bursal disease in poultry: a review. Avian Pathol. 29, 175-194. Birghan, C., Mundt, E., Gorbalenya, A.E., 2000. A non-canonical lon proteinase lacking the ATPase domain employs the ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus. EMBO J. 19, 114-123.
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Boot, H.J., ter Huurne, A.A., Hoekman, A.J., Peeters, B.P., Gielkens, A.L., 2000. Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype. J. Virol. 74, 6701-6711.
Brandt, M., Yao, K., Liu, M., Heckert, R.A., Vakharia, V.N., 2001. Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus. J. Virol. 75, 11974-11982.
-p
Brown, M.D., Skinner, M.A., 1996. Coding sequences of both genome segments of a European 'very virulent' infectious bursal disease virus. Virus Res. 40, 1-15.
Chen, G., He, X., Yang, L., Wei, P., 2018. Antigenicity characterization of four representative natural
re
reassortment IBDVs isolated from commercial three-yellow chickens from Southern China reveals different subtypes co-prevalent in the field. Vet. Microbiol. 219, 183-189. Vet. Rec. 125, 271-272.
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Chettle, N., Stuart, J.C., Wyeth, P.J., 1989. Outbreak of virulent infectious bursal disease in East Anglia. Cosgrove, A.S., 1962. An Apparently New Disease of Chickens: Avian Nephrosis. Avian Dis. 6, 385-389.
na
Coulibaly, F., Chevalier, C., Gutsche, I., Pous, J., Navaza, J., Bressanelli, S., Delmas, B., Rey, F.A., 2005. The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120, 761-772.
Escaffre, O., Le Nouen, C., Amelot, M., Ambroggio, X., Ogden, K.M., Guionie, O., Toquin, D., Muller,
ur
H., Islam, M.R., Eterradossi, N., 2013. Both genome segments contribute to the pathogenicity of very virulent infectious bursal disease virus. J. Virol. 87, 2767-2780. Eterradossi, N., Picault, J.P., Drouin, P., Guittet, M., L'Hospitalier, R., Bennejean, G., 1992.
Jo
Pathogenicity and preliminary antigenic characterization of six infectious bursal disease virus strains isolated in France from acute outbreaks. Zentralbl. Veterinarmed. B. 39, 683-691.
Fahey, K.J., Erny, K., Crooks, J., 1989. A conformational immunogen on VP-2 of infectious bursal disease virus that induces virus-neutralizing antibodies that passively protect chickens. J. Gen. Virol. 70 ( Pt 6), 1473-1481. Fan, L., Wu, T., Hussain, A., Gao, Y., Zeng, X., Wang, Y., Gao, L., Li, K., Wang, Y., Liu, C., Cui, H., Pan, Q., Zhang, Y., Liu, Y., He, H., Wang, X., Qi, X., 2019. Novel variant strains of infectious bursal disease virus isolated in China. Vet. Microbiol. 230, 212-220. Gao, L., Li, K., Qi, X., Gao, H., Gao, Y., Qin, L., Wang, Y., Shen, N., Kong, X., Wang, X., 2014. Triplet 11
amino acids located at positions 145/146/147 of the RNA polymerase of very virulent infectious bursal disease virus contribute to viral virulence. J. Gen. Virol. 95, 888-897. Garriga, D., Querol-Audi, J., Abaitua, F., Saugar, I., Pous, J., Verdaguer, N., Caston, J.R., Rodriguez, J.F., 2006. The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid. J. Virol. 80, 6895-6905. Icard, A.H., Sellers, H.S., Mundt, E., 2008. Detection of infectious bursal disease virus isolates with unknown antigenic properties by reverse genetics. Avian Dis. 52, 590-598. Jackwood, D.H., Saif, Y.M., 1987. Antigenic diversity of infectious bursal disease viruses. Avian Dis. 31, 766-770. Jackwood, D.J., 2012. Molecular epidemiologic evidence of homologous recombination in infectious bursal disease viruses. Avian Dis. 56, 574-577. Jackwood, D.J., 2017. Advances in vaccine research against economically important viral diseases of food animals: Infectious bursal disease virus. Vet. Microbiol. 206, 121-125.
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Jackwood, D.J., Cookson, K.C., Sommer-Wagner, S.E., Le Galludec, H., de Wit, J.J., 2006. Molecular characteristics of infectious bursal disease viruses from asymptomatic broiler flocks in Europe. Avian Dis. 50, 532-536.
Jackwood, D.J., Sommer-Wagner, S.E., 2005. Molecular epidemiology of infectious bursal disease
viruses: distribution and genetic analysis of newly emerging viruses in the United States. Avian
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Dis. 49, 220-226.
Jackwood, D.J., Sommer-Wagner, S.E., 2011. Amino acids contributing to antigenic drift in the infectious bursal disease Birnavirus (IBDV). Virology 409, 33-37.
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Jackwood, D.J., Sreedevi, B., LeFever, L.J., Sommer-Wagner, S.E., 2008. Studies on naturally occurring infectious bursal disease viruses suggest that a single amino acid substitution at position 253 in VP2 increases pathogenicity. Virology 377, 110-116.
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Kurukulsuriya, S., Ahmed, K.A., Ojkic, D., Gunawardana, T., Gupta, A., Goonewardene, K., Karunaratne, R., Popowich, S., Willson, P., Tikoo, S.K., Gomis, S., 2016. Circulating strains of variant infectious bursal disease virus may pose a challenge for antibiotic-free chicken farming in Canada. Res.Vet. Sci. 108, 54-59.
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Ladman, B.S., Loupos, A.B., Gelb, J., Jr., 2006. Infectious bronchitis virus S1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization. Avian Pathol. 35, 127-133.
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Lee, C.C., Ko, T.P., Chou, C.C., Yoshimura, M., Doong, S.R., Wang, M.Y., Wang, A.H., 2006. Crystal structure of infectious bursal disease virus VP2 subviral particle at 2.6A resolution: implications in virion assembly and immunogenicity. J. Struct. Biol. 155, 74-86.
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Letzel, T., Coulibaly, F., Rey, F.A., Delmas, B., Jagt, E., van Loon, A.A., Mundt, E., 2007. Molecular and structural bases for the antigenicity of VP2 of infectious bursal disease virus. J. Virol. 81, 12827-12835.
Lu, Z., Zhang, L., Wang, N., Chen, Y., Gao, L., Wang, Y., Gao, H., Gao, Y., Li, K., Qi, X., Wang, X., 2015. Naturally occurring reassortant infectious bursal disease virus in northern China. Virus Res. 203, 92-95. Lucio, B., Hitchner, S.B., 1979. Response of susceptible versus immune chicks to killed, live-modified, and wild infectious bursal disease virus vaccines. Avian Dis. 23, 1037-1050. Luque, D., Rivas, G., Alfonso, C., Carrascosa, J.L., Rodriguez, J.F., Caston, J.R., 2009. Infectious bursal disease virus is an icosahedral polyploid dsRNA virus. Proc. Natl. Acad. Sci. U S A. 106, 12
2148-2152. Müller, H., Islam, M.R., Raue, R., 2003. Research on infectious bursal disease—the past, the present and the future. Vet. Microbiol. 97, 153-165. Perozo, F., Villegas, A.P., Fernandez, R., Cruz, J., Pritchard, N., 2009. Efficacy of single dose recombinant herpesvirus of turkey infectious bursal disease virus (IBDV) vaccination against a variant IBDV strain. Avian Dis. 53, 624-628. Qi, X., Gao, H., Gao, Y., Qin, L., Wang, Y., Gao, L., Wang, X., 2009. Naturally occurring mutations at residues 253 and 284 in VP2 contribute to the cell tropism and virulence of very virulent infectious bursal disease virus. Antiviral Res. 84, 225-233. Qi, X., Gao, L., Qin, L., Deng, X., Wu, G., Zhang, L., Fei, Y., Ren, X., Gao, Y., Gao, H., Wang, Y., Wang, X., 2011. Genomic Sequencing and Molecular Characteristics of A Very Virulent Strain of Infectious Bursal Disease Virus Isolated in China. Agric. Sci. Technol. 16, 1565-1569. Qi, X., Gao, X., Lu, Z., Zhang, L., Wang, Y., Gao, L., Gao, Y., Li, K., Gao, H., Liu, C., Cui, H., Zhang,
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Y., Wang, X., 2016. A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus. Sci. China Life Sci. 59, 717-723.
Qi, X., Qin, L., Gao, Y., Gao, H., Li, Y., Li, G., Lu, Z., Wang, N., Chen, Y., Zhang, L., Li, K., Wang, Y., Wang, X., 2015. Genetic Analysis of the VP2 Hypervariable Region of Thirty-six Infectious
Bursal Disease Virus Isolates in China during 2009-2012. Agric. Sci. Technol. 12, 1950-1953.
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Qi, X., Zhang, L., Chen, Y., Gao, L., Wu, G., Qin, L., Wang, Y., Ren, X., Gao, Y., Gao, H., Wang, X.,
2013. Mutations of residues 249 and 256 in VP2 are involved in the replication and virulence of infectious Bursal disease virus. PloS one 8, e70982.
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Stoute, S.T., Jackwood, D.J., Crossley, B.M., Michel, L.O., Blakey, J.R., 2019. Molecular epidemiology of endemic and very virulent infectious bursal disease virus genogroups in backyard chickens in California, 2009-2017. J. Vet. Diagn. Invest. 31, 371-377.
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Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729. Vakharia, V.N., He, J., Ahamed, B., Snyder, D.B., 1994. Molecular basis of antigenic variation in infectious bursal disease virus. Virus Res. 31, 265-273.
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van den Berg, T.P., Eterradossi, N., Toquin, D., Meulemans, G., 2000. Infectious bursal disease (Gumboro disease). Rev. Sci. Tech. 19, 509-543. Wang, X.M., Zeng, X.W., Gao, H.L., Fu, C.Y., Wei, P., 2004. Changes in VP2 gene during the
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attenuation of very virulent infectious bursal disease virus strain Gx isolated in China. Avian Dis. 48, 77-83.
Yu, F., Ren, X., Wang, Y., Qi, X., Song, J., Gao, Y., Qin, L., Gao, H., Wang, X., 2013. A single amino
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acid V4I substitution in VP1 attenuates virulence of very virulent infectious bursal disease virus (vvIBDV) in SPF chickens and increases replication in CEF cells. Virology 440, 204-209. Zachar, T., Popowich, S., Goodhope, B., Knezacek, T., Ojkic, D., Willson, P., Ahmed, K.A., Gomis, S., 2016. A 5-year study of the incidence and economic impact of variant infectious bursal disease viruses on broiler production in Saskatchewan, Canada. Can. J. Vet. Res. 80, 255-261.
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Figure legends
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Fig. 1. Immunoprotective evaluation of vvIBDV vaccines against novel variant IBDV strain SHG19 using specific-pathogen-free (SPF) Chickens. A. IBDV-specific antibody induced by three types of vvIBDV vaccines prior to challenge. B. The bursa:body weight index (BBIX) at 5 and 10 days post-infection (d p.i.). C. The spleen/body weight ratio. D. The bursae of different groups. E. The histopathological appearance of the bursal sections (hematoxylin and eosin staining). The mean titers and standard deviations (error bars) from six (A) or three (B, C) independent samples are shown. ∗ and ∗∗ represent P < 0.05 and P < 0.01 compared with PBS (B) group or control group (C).
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Fig. 2. Phylogenetic tree analysis of amino acid sequences of the viral polyprotein (A) and VP1 (B). The trees were generated by the neighbor-joining method using MEGA6 with 1000 replications. The novel variant IBDV strain SHG19 is highlighted with the solid circular.
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Fig. 3. Characteristic amino acid substitutions in VP5, polyprotein, and VP1 of variant IBDV and vvIBDV. Var, variant; VV, very virulent; AT, attenuated strain. Asterisks indicate residues identical to the sequence of novel variant IBDV strain SHG19.
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Table 1 Reference IBDV strains. Phenotype
OKYM HK46 D6948 KS UK661 89163 Gx HLJ0504 HuB-1 BD399 Variant E 9109
Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Very virulent Variant Variant
GenBank accession no.
IBDV Strains
Segment A
Segment B
D49706 AF092943 AF240686 DQ927042 NC-004178 HG974563 AY444873 GQ451330 KF569805 AF362776 AF133904 AY462027
D49707 AF092944 AF240687 DQ927043 NC-004179 HG974564 AY705393 GQ451331 GQ449693 AF362770 AF133905 AY459321
Phenotype
IM F52/70 CU-1 Gt CT P2 NB CEF-94 HZ2 D78 JD1 OH
Classic Classic Attenuated Attenuated Attenuated Attenuated Attenuated Attenuated Attenuated Attenuated
GenBank accession no. Segment A
Segment B
AY029166 HG974565 X16107 DQ403248 AJ310185 X84034 AY319768 AF133904 AF321054 AF499929 AF321055 U30818
AY029165 HG974566 AF362775 DQ403249 AJ310186 X84035 AY654284 AF133905 AF493979 AF499930 AY103464 U30819
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IBDV Strains
Attenuated
Serotype II
1-6H-3A
2-3B-5D
89163
+ +
+ +
+ +
SHG19
—
—
—
+ + —
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HLJ0504
2-5C-6F
7D4
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1-2C-7C
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Table 2 Comparison of MAb reactivity pattern of novel variant IBDV strain SHG19 and vvIBDV.
+ + +
8G
4-5D-2E
3-10H-7A
+ +
+ +
+ +
—
—
—
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Note: “+” and “-” indicate the positive and negative reaction signal of the immunofluorescence assays.
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Table 3 R value of cross-neutralization assays. SHG19 Gx HLJ0504 SHG19 1.00 Gx 0.64 1.00 HLJ0504 0.35 1.05 1.00
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