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Sequence variations of the MHC class I gene exon 2 and exon 3 between infected and uninfected chickens challenged with Marek’s disease virus
Infection, Genetics and Evolution 21 (2014) 103–109
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Sequence variations of the MHC class I gene exon 2 and exon 3 between infected and uninfected chickens challenged with Marek’s disease virus Ye Wang a,1, Mohan Qiu b,1, Jiandong Yang c, Xiaoling Zhao a, Yan Wang a, Qing Zhu a, Yiping Liu a,⇑ a
Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Chengdu Campus, Chengdu, Sichuan 611130, China Sichuan Animal Science Academy, Chengdu, Sichuan 610066, China c College of Animal Science and Technology, Sichuan Agricultural University, Ya’an, Sichuan 625014, China b
a r t i c l e
i n f o
Article history: Received 23 May 2013 Received in revised form 21 October 2013 Accepted 24 October 2013 Available online 4 November 2013 Keywords: Chicken MDV challenge MHC class I exon 2 Exon 3 Evolutionary differences
a b s t r a c t The major histocompatibility complex (MHC) among chickens has been well established as being associated with disease resistance and pathogens infection, but the genetic differences in MHC between chickens susceptible to certain infections and those chickens that remain uninfected have not been sufficiently determined. In this study, we sought the genetic basis that may underlie differences in susceptibility to infection among chickens by challenging four groups of broilers with Marek’s disease virus (MDV). Over the course of the experiment, lesions began to appear between 21 and 35 days post challenge (dpc), and commercial broilers were not necessarily better than indigenous chickens in terms of disease resistance. The four groups showed neutral resistance to MDV infection validated by challenge results and evolutionary analysis of exons 2 and 3 of the MHC class I region. Several variable sites in exon 2 and exon 3 were exclusively appeared in infected chickens. Exon 3 was likely more crucial than exon 2 in disease resistance. Our observations offered a support for a potential association between promiscuous pathogens and conspicuous genetic diversity in the MHC class I region. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The chicken major histocompatibility complex (MHC) is recognized as the most polymorphic coding region known in the vertebrate genome, with strong associations to both disease resistance and pathogen infection (Chaves et al., 2010; Eimes et al., 2010; Fulton et al., 2006). The MHC itself is comprised of two genes (MHC class I and class II) and encodes membrane glycoprotein receptors that present foreign peptides for T cells – likely a critical mechanism in scheduling immune responses (Silva and Edwards, 2009). Though intriguing for the sheer complexity of the MHC’s polymorphic nature, gaining a clearer understanding of the precise function of the MHC has broad implications. Marek’s disease (MD), for example, is a highly contagious T-cell lymphoma caused by Marek’s disease virus (MDV) that causes significant economic losses in the production of poultry. A prior study on MDV indicated that the genes associated with the B complex involved in resistance to MD are localized within the B–F/B–L region (Hepkema et al., 1993), but a more recent and clearer picture indicated that MDV is capable of down-regulating ⇑ Corresponding author. Address: Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Chengdu Campus, 211# Huimin Road, Wenjiang, Sichuan 611130, China. Tel./fax: +86 28 86290987. E-mail address: [email protected] (Y. Liu). 1 These authors contributed equally to this work, and shall share the first author. 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.10.020
the surface expression of the MHC class I gene in the immediate early phase, a process completed by the early phase of viral gene expression (Hunt et al., 2001). Similarly, recent studies validated the differences of class I expression between both MD-resistant and -susceptible chickens (Dalgaard et al., 2003, 2009). The currently verified ‘‘minimal essential MHC’’ hypothesis suggests that in chickens, the abundant class I alleles result in either the resistance or susceptibility to specific pathogens (Kim et al., 2008; Owen et al., 2008). The Class I molecule itself consists of one alpha chain and b2-microglobuin, of which, the alpha chain is the transmembrane tail which is composed by domains a1, a2, a3 (Jin et al., 2010). Indeed, a1 and a2 are the more important domains that assist in the formation of the residues present in antigen bonding region, which are encoded by the exons 2 and 3 of class I, respectively (Cloutier et al., 2011). Accordingly, the exon 2 and 3 regions would suffer from most pathogen-driven forces and selection pressures, which may explain the previously observed accumulation of diversity and polymorphisms as well as the remarkable complexity of the chicken MHC (Bumstead and Kaufman, 2004; Piertney and Oliver, 2005). Among chickens, serological studies have identified MD-resistant and -susceptible haplotypes, such as B21 and B19, but little has been done to explain the actual molecular differences of key regions of the MHC class I between chickens that are either MD-resistant or -susceptible. In this study, we opted to examine the underlying genetic basis that may help explain resistance or susceptibility to MD by
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challenging four groups of chickens with a very virulent (vv) MDV before sequencing exons 2 and 3 of the MHC class I gene in order to (a) screen the sequence variations across four chicken groups, and (b) to investigate whether the resulting infected or uninfected chickens were under diverse selection pressures or had any significant differences between them. 2. Materials and methods
Dalian, China). A standard HotStart PCR process was used for PCR amplification, which is comprised of an initial denaturation cycle of 5 min at 94 °C, 35 cycles at 94 °C for 45 s, annealing at 62 °C (for exon 2) and 58.8 °C (for exon 3) for 45 s, and 72 °C for 50 s, followed by a final extension cycle at 72 °C for 8 min, ending with an incubation at 4 °C. PCR products were checked on 1% agarose gel, following which all PCR products were purified on spin columns and sequenced by Invitrogen Corporation Shanghai (Shanghai, China).
2.1. Chickens 2.5. Data analysis All procedures used in this study were in accordance with the guide for the care and use of laboratory animals. In brief, we collected 193 25-day old broiler chickens for challenging with MDV from four groups: 49 Huiyang chickens – famous Chinese indigenous chickens known for their meat quality; 44 AA broilers from commercial lines; and 50 A03 chickens and 50 E1 chickens from specialized lines, which were bred and maintained at the Institute of Animal Science, Guangdong Academy of Agricultural Sciences. Prior to challenge with MDV, all chickens were tested to ensure they were specific-pathogen-free (SPF), i.e., free from any antibodies to MDV, Newcastle disease virus, reovirus, chicken anaemia virus (CAV), infectious bursal disease virus (IBDV). All chickens were also kept in isolators drinking ad libitum with commercial food, to maintain integrity of the experiment. 2.2. MDV challenge and postmortem Blood samples were taken from each individual prior to the MDV challenge, and chickens were challenged by intra-abdominal injection with the very virulent virus MDV J-1 strain, which is a virulent reference strain in China (provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences), at a dosage of 1000 PFU (plaque forming unit) per chicken in 0.2 ml of diluent. Clinical symptoms and mortality were observed daily for each individual. At 21 days post challenge (dpc), we began euthanizing chickens, 10 birds per group each week, until at 41 dpc all remaining birds were sacrificed for postmortem analysis. In the interim, we conducted executed postmortem examination for chickens either dying or which were euthanized to examine gross MD tumor lesions. 2.3. Genomic DNA extraction and primer design Following MDV challenge, chickens from the four groups (Huiyang, AA broilers, A03 and E1 lines) that had either became infected or remained uninfected were randomly chosen and placed into two groups for sequencing (Table S1) of either exon 2 (44 individuals) or exon 3 (47 individuals). Genomic DNA was extracted from whole blood by the standard phenol/chloroform method. Two primer pairs (primer pair 1, F: 50 -ccgcccgtaaccccaccc-30 /R: 50 -agcccatcccacacccacgg-30 ; primer pair 2, F: 50 -cgcggctccgtgggtgtgg-30 / R: 50 -cgcggctccgtgggtgtgg-30 ) were designed according to the chicken reference sequence (GenBank accession no. M31012) to amplify exons 2 (396 bp fragment) and 3 (501 bp fragment), respectively. Primer pairs were synthesized by Invitrogen Corporation Shanghai (Shanghai, China). 2.4. PCR amplification and sequencing PCR amplification was performed in a 50 lL reaction mixture containing 100 ng of DNA, 10 mmol/L Tris–HCl (pH 8.3), 2.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mM of each dNTPs, 10 pmol/L of each primer, and 1 unit of Taq polymerase (Takara,
In total, 44 individuals were sequenced for exon 2 and 47 individuals were sequenced for exon 3. Sequences were edited and assembled using BioEdit 7.1.11 (Hall, 1999). For comprehensive analysis, 21 exon 2 sequences from GenBank were included, which were composed of 5 White leghorn chickens (WL) (accession numbers EU747296.1, EU747294.1, EU747292.1, EU747293.1, EU747295.1), 8 Beijing You chickens (BJU) (accession numbers EU755019.1, EU755017.1, EU755015.1, EU755013.1, EU755011.1, EU755020.1, EU755018.1, EU755016.1), 8 blue-egg chickens (accession numbers AY489145.1, AY489147.1, AY489149.1, AY489151.1, AY489155.1, AY489157.1, AY489159.1, AY489153.1). Similarly, 15 exon 3 sequences from GenBank were included for the analysis, which were composed of 4 HH line chickens (accession numbers AY725441.1, AY725439.1, AY725445.1, AY725447.1), 3 LL line chickens (accession numbers AY725453.1, AY725451.1, AY725449.1), 8 blue-egg chickens (accession numbers AY489145.1, AY489147.1, AY489149.1, AY489151.1, AY489155.1, AY489157.1, AY489159.1, AY489153.1). 13 sequences from GenBank were used in the analysis of both exon 2 and exon 3, which were composed of 7 B21 haplotype sequences (accession numbers S78682, AY234769, AF231499, AF231503, AF231502, AF231500 and AF231501), 2 B19 haplotype sequences (accession numbers Z54317 and Z54318), 1 B2 haplotype sequence (accession number AB426141), 1 B6 haplotype sequence (accession number Z54325), 1 B12 haplotype sequence (accession number Z54326) and sequence M31012.1. MEGA 5.1 (Tamura et al., 2011) was used to perform multiple sequence alignment and the generation of amino acid sequences. DnaSP 4.50.3 (Librado and Rozas, 2009) was used to analyze the number of haplotypes (h), haplotype diversity (Hd), nucleotide diversity (p), the average number of nucleotide differences (K). The numbers of synonymous (dS) and non- synonymous (dN) substitutions per site were also estimated. Phylogenetic trees were reconstructed using the neighbor joining (NJ) method (Saitou and Nei, 1987), and the MHC haplotypes B21 and B19 were taken into consideration to assist in judging the relative resistance and susceptibility of the four groups of chickens. 3. Results 3.1. Incidence of Marek’s disease after challenge Excluding four chickens that died during the course of the study, either without artificial interference or from MDV infection, the remaining chickens were executed on a precise schedule as stated earlier, leaving 19.2% of total chickens (37/193) significantly infected with MDV (Table 1). The incidence of MDV infection during each period of time for each breed is shown in Table 1. Overall, A03 chickens had the highest morbidity ratio (28.6%) while the AA chicken had the lowest ratio (11.6%), with the susceptibility order of the four lines as A03, E1, Huiyang, and AA. The morbidity of MD mainly occurred between 21 dpc and 35 dpc. No infected chicken was found at 42 dpc or even at 35 dpc in the E1 population.
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Y. Wang et al. / Infection, Genetics and Evolution 21 (2014) 103–109 Table 1 Morbidity and mortality rates at various time intervals following MDV challenge among four chicken lines. Chicken lines
N
AA A03 E1 Huiyang
43 49 48 49
Tumor incidence at different intervals 21 dpc
28 dpc
35 dpc
42 dpc
20% 70% 60% 50%
10% 40% 40% 30%
20% (2/10) 20% (2/10) 0% (0/10) 10% (1/10)
0% 0% 0% 0%
(2/10) (7/10) (6/10) (5/10)
(1/10) (4/10) (4/10) (3/10)
(0/13) (0/18) (0/17) (0/18)
Morbidity rate
Mortality rate
11.6% 28.6% 22.9% 20.4%
– 7.1% (1/14) 9.1% (1/11) 10% (1/10)
(5/43) (14/49) (11/48) (10/49)
N is the valid number of chickens that died either due to MDV challenge or human interference. ‘‘–’’ denotes no chicken died during the course of challenge.
3.2. Variation and high levels of diversity of exon 2
whole population were 0.04002 and 10.924, respectively. Among the four groups, A03 had the highest nucleotide diversity (0.03914) and nucleotide differences (10.684), E1 was next to A03 (p = 0.03810; K = 10.400), and Huiyang chickens had the lowest nucleotide diversity (0.02821) and nucleotide differences (7.700). Theta values were 0.02911, 0.03983, 0.03627 and 0.02989 for AA, A03, E1 and Huiyang, respectively, yielding an average value of 0.03815. The Tajima’s D test had a positive value of 0.23314 in E1 population, while the other three populations were negative (AA, 0.10613; A03, 0.06954; Huiyang, 0.41429) (Table 2).
A total of 77 sequences (including 33 reported sequences; with a truncated length of 265 bp) were analyzed for exon 2. There was no length variation except for a base insertion at the 100th position for individual C195 (Table S2). Sixty-nine variable sites were found, yielding a mean nucleotide mutation rate of 26.04% (69/265). Among these variable sites, 16 were singleton variants, 53 were parsimony informative sites. Interestingly, variations at sites 12 (C > A), 195 (C > G), 205 (A > C), and 237 (A > T) appeared exclusively to infected chickens. Analysis of exon 2 showed high levels of haplotype diversity (H) among the four groups, with values of 0.985 for AA, 0.994 for A03, 1.000 for E1, and 1.000 for Huiyang chicken (Table 2). High levels of haplotype diversity indicated abundant nucleotide diversity, in this case AA had the highest p (0.04477) and K values (11.818), whereas A03 had the lowest values for both p (0.02924) and K (7.719) among the four populations. Theta-W and Tajima’s D were also calculated for each of the four groups to further clarify these initial observations. The polymorphism index of Theta values were 0.04641, 0.03035, 0.03080 and 0.04040 for AA, A03, E1 and Huiyang, respectively. The Huiyang chicken and E1 chicken had a positive Tajima’s D value, while populations AA (0.16084) and A03 (0.14386) had a negative value (Table 2). There was no parsimony informative site in the Huiyang chickens, perhaps due to its modest sample size.
3.4. Different selection pressures between infected and uninfected chickens As the antigen bonding region, the a1 and a2 domains encoded by exons 2 and 3 might have undergone the strongest pathogen selection. We calculated the values of dN/dS and executed a codon-based Z test to determine whether there was a selection in those two regions (Table 3). The peptide binding region (PBR) was inferred and superimposed on sequences using previously published data from a great reed warbler (Richardson and Westerdahl, 2003; Westerdahl et al., 2000). We found no significant selection in exon 2, while the PBR had a higher dN/dS value (2.017, P = 0.97) than non-PBR (0.979, P = 0.48). The uninfected groups generally had higher dN/dS values than infected ones. However, analysis did show that the PBR of exon 3 was under significant balancing selection (4.356, P = 0.02) while the non-PBR of exon 3 were under an unapparent purifying selection (0.501, P = 0.09). Interestingly, in the PBR of uninfected chickens, the dN/dS value was significant (7.253, P = 0.01) and much higher than that found among the infected chickens (2.400, P = 0.05), while this was not found in either the non-PBR or the other remaining sites. Compared to exon 2, exon 3 also had a lower dN/dS value (PBR: 1.123, P = 0.35; nonPBR: 0.531, P = 0.08), suggesting that exon 2 might suffer balancing selection while exon 3 was under purifying selection.
3.3. Variation and high levels of diversity of exon 3 Analysis of 75 sequences (including 28 reported sequences) of exon 3 yielded a total of 55 variable sites along the 273 bp fragment (Table S3). Of the 55 variable sites, six were singleton variable sites and 49 were parsimony informative sites. Intriguingly, variable sites 11, 178, 199 were found only in infected group. Exon 3 showed a markedly high level of haplotype diversity, with an average of haplotype diversity (H) 0.998 (Table 2). The overall nucleotide diversity and nucleotide differences of the
Table 2 Nucleotide diversity of MHC class I exon 2 and exon 3 among four chicken populations. Chicken lines Exon 2 AA A03 E1 Huiyang Total Exon 3 AA A03 E1 Huiyang Total
N
No. of polymorphic sites
No. of parsimony informative sites
K
p
Theta (per site) from Eta
Tajima’s D
Significance
12 19 10 3 44
34 26 23 14 38
25 25 18 0 31
11.818 7.719 8.422 10.000 9.795
0.04477 ± 0.00746 0.02924 ± 0.00514 0.03190 ± 0.00762 0.03788 ± 0.01045 0.03710 ± 0.00368
0.04641 0.03035 0.03080 0.04040 0.04006
0.16084 0.14386 0.17025 0.25784
P > 0.10 P > 0.10 P > 0.10 – P > 0.10
12 19 11 5 47
21 31 27 16 39
14 23 20 5 31
7.758 10.684 10.400 7.700 10.924
0.02842 ± 0.00259 0.03914 ± 0.00303 0.03810 ± 0.00405 0.02821 ± 0.00790 0.04002 ± 0.00204
0.02911 0.03983 0.03627 0.02989 0.03815
0.10613 0.06954 0.23314 0.41429 0.16933
P > 0.10 P > 0.10 P > 0.10 P > 0.10 P > 0.10
K is average number of nucleotide difference; p is average number of nucleotide diversity.
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Table 3 Estimates of average codon-based evolutionary divergence and positive selection in chickens challenged by MDV. Region
Infection status
PBR
Infected Uninfected Overall Infected Uninfected Overall Infected Uninfected Overall
Non-PBR
All sites
Exon 2
Exon 3
dN (±SE)
dS (±SE)
dN/dS
p
dN (±SE)
dS (±SE)
dN/dS
p
0.0650 ± 0.0196 0.0887 ± 0.0323 0.0823 ± 0.0030 0.0283 ± 0.0093 0.0305 ± 0.0094 0.0297 ± 0.0088 0.0361 ± 0.0009 0.0424 ± 0.0103 0.0412 ± 0.0098
0.0415 ± 0.0428 0.0408 ± 0.0402 0.0407 ± 0.0388 0.0294 ± 0.0137 0.0203 ± 0.0100 0.0303 ± 0.0136 0.0373 ± 0.0125 0.0358 ± 0.0130 0.0367 ± 0.0125
1.568 2.177 2.017 0.963 1.503 0.979 0.968 1.187 1.123
0.28 0.14 0.16 0.46 0.21 0.48 0.46 0.28 0.35
0.0773 ± 0.0322 0.0683 ± 0.0275 0.0706 ± 0.0283 0.0302 ± 0.01172 0.0283 ± 0.0105 0.0270 ± 0.0101 0.0360 ± 0.0108 0.0284 ± 0.0078 0.0284 ± 0.0079
0.0274 ± 0.0265 0.0094 ± 0.0093 0.0162 ± 0.0162 0.0569 ± 0.0205 0.0531 ± 0.0183 0.0539 ± 0.0200 0.0552 ± 0.0177 0.0540 ± 0.0174 0.0535 ± 0.0017
2.400 7.253 4.356 0.532 0.533 0.501 0.651 0.525 0.531
0.05 0.01 0.02 0.09 0.09 0.09 0.15 0.08 0.08
Note: analyses of the numbers of synonymous (dS) and non-synonymous (dN) substitutions per site were conducted using the Nei–Gojobori method in MEGA 5.1. Standard error estimates are obtained by a bootstrap procedure (2000 replicates). All positions containing alignment gaps and missing data were eliminated. p Values were calculated by the codon-based Z test with the alternative hypothesis of dN > dS of dN < dS. PBR, peptide binding region; Non-PBR, region beyond the peptide binding region.
3.5. Phylogenetic trees of exon 2 and exon 3 The unrooted NJ tree for exon 2 among the four groups, along with the aforementioned sequences from GenBank, had a complex clustering pattern (Fig. 1). Chickens from the four groups were intermixed together with a blear divergence. There was no significant clustering between infected individuals and uninfected ones. This clustering pattern hardly changed when haplotypes B6 and B12 were included in the analysis. B2 was clustered with one blue-egg chicken in either exon 2 or exon 3. Likewise, WL, BJY and blue-egg caipira groups were apparently clustered together, most blue-egg caipiras were clustered together with haplotype B21, and WL and BJY were mixed with each other. Similarly, for exon 3, no significant clustering differences were found between infected individuals and uninfected chickens (Fig. 2). The complicated phylogenetic tree of exon 3 showed that the seven lines were permeated with each other. 4. Discussion A variety of studies have demonstrated that the chicken MHC gene was extensively associated with pathogens infection like avian influenza (AI), Newcastle disease (ND), Marek’s disease virus (MDV) (Abdul-Careem et al., 2008; Liu et al., 2009; Niikura et al., 2007; Schou et al., 2010; Shiina et al., 2007). This finding is consistent with the basic premises of an ongoing ‘‘evolutionary arms race’’ between the large numbers of pathogens in a given environment and the MHC gene of given hosts gave rise to the observed high genetic diversity of the MHC, which was presumed to be maintained via balancing selection (Borghans et al., 2004). In this study, we conducted a MDV challenge on four chicken groups (AA, A03, E1, Huiyang) followed by genetic sequencing of the MHC gene. We further used available data from WL, BJY, blueegg caipira, HH and LL chicken lines to construct a sequence profile of exons 2 and 3 of the MHC class I gene in order to gain a more complete picture of sequence diversity, balancing selection and phylogenetic tree of the chickens challenged with MDV. While the low MDV infection ratio and limited experimental capacity prevent more practical samples apply to our study, further investigation would be helpful to solidify our current observation. 4.1. Infection characteristic of MDV challenge MDV challenge resulted in the first death of a chicken with MD lesions at 20 dpc, with the incidence of lesions centrally appearing sometime between 21 dpc and 35 dpc, which were consistent with previous findings (Abdul-Careem et al., 2009; Sharma and Stone, 1972). In general, the morbidity of MD lesions was 19.2% (37/
193), though this was relatively lower than the rate found among the chickens with B19 haplotypes but higher than the chickens with B21 haplotypes (Dalgaard et al., 2003). Though in no way a conclusive finding, this result offers some intriguing circumstantial evidence that supports our findings on phylogeny clustering. One cautionary note is worth mentioning, these results could also support an inference that indigenous chicken breeds may not possessed better behavior to disease resistance than commercial chicken breeds, as the AA broilers had a striking lower morbidity and mortality than our local breed Huiyang chicken. This could instead be explained by gene exchange and heterosis, which has improved the disease resistance of chickens without fundamentally altering the composition of the MHC. 4.2. Polymorphisms of exons 2 and 3 of the MHC class I indicated promiscuous peptide bonding Many variable sites were found both in exon 2 (18.94%) and exon 3 (16.12%) with an uneven distribution, which were retrenched to 202–249 sites and 102–199 sites, respectively. Transferred into amino acid sequences, over half of the peptide bonding sites (PBS) were variable for exon 2 (58.8%), with slightly less in exon 3 (46.7%). Those highly variable regions may have led to a high resolution to antigens and help in recognizing novel pathogens to ensure rapid adaptation to environmental changes (Schut et al., 2011a). Moreover, this inference has been verified in human HLA class I gene, in which positive correlations were identified between HLA genetic diversity and pathogen richness (Prugnolle et al., 2005). Similarly, host-pathogen co-evolution has been purported to be the key factor of MHC polymorphism (Borghans et al., 2004). Among the four groups (AA, A03, E1, Huiyang), AA was the most variable group while A03 was the least diverse group concerning the exon 2 variation. As for exon 3, A03 was the most variable group, and Huiyang was the least varied. This is largely consistent with the realities of the modern poultry industry. To cater to the special market requirements, multiple chicken lines were utilized to construct the commercial chicken production system, which resulted in a potentially unintended consequence of leading to complex genetic variation among commercial chickens (Emmerson, 1997). This may explain the pattern for high polymorphisms of both the AA and A03 commercial lines. The diverse pattern for exon 2 and exon 3 suggests that the two exons might perform distinctly different functions during the antigen bonding process, though the actual physical distance between exons 2 and 3 was relatively compact. The binding groove of MHC class I, however, has an unusually large central cavity, which confers substantial conformational flexibility to the crucial residue Arg 9, which then in turn allows the remodeling of key peptide-binding sites (Spurgin
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Fig. 1. Phylogenetic tree of exon 2 of MHC class I sequences. 77 sequences (including 33 reported sequences) were used to reconstruct the NJ tree. IDs marked with blue dots are chickens infected with MDV. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and Richardson, 2010). Taken together, the aforementioned evidence and the high levels of polymorphism do offer some insights that support a potential association between promiscuous pathogens and conspicuous genetic diversity. 4.3. Balance selection was acted on a3 domain Given our current knowledge on MHC and the role pathogens play in evolution, it was reasonable to expect that pathogen driven selections caused by MDV occurred during chickens’ evolution history (Spurgin and Richardson, 2010), and that in turn balancing selection acted as the major mechanism to maintain variations of MHC genes (Hedrick, 1998). In this study, we calculated dN/dS value of exon 2 and exon 3 between both infected and uninfected groups using the PBR and non-PBR regions to identify whether the a1 and a2 domains were under balancing selection or not, and to check whether the differential selection pressure between infected and uninfected chickens exists. Balancing selection was significantly
found in the PBR of exon 3 and PBR of exon 2 without predominance (Table 3). Of all the PBRs, 10/17 of exon 2, and 7/15 of exon 3 were variable, which suggested that only sites with an immediate association to pathogens could be placed under acute selection pressure. These results and the subsequent inference on pathogens and selection pressure are consistent with previous studies on blue tits (Cyanistes caeruleus) (Schut et al., 2011a) and red-billed gulls (Larus scopulinus) (Cloutier et al., 2011). Collectively, the above findings and the previous researches implicate a preference for balancing selection on sites that immediately contribute to antigen bonding, as opposed to other conserved sites. Rather intriguingly, nearly all uninfected groups had a higher dN/dS value than infected groups, whether in terms of PBR sites, non-PBR sites, or all sites in general. This discrepancy may be the result of intense purifying selection caused by MDV mortality. Pathogens are a key environmental factor in reducing or controlling the adaptation of animals, which would in turn induce purifying selection and play a important role in creating population
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Fig. 2. Phylogenetic tree of exon 3 of MHC class I sequences. 75 sequences (including 28 reported sequences) were used to reconstruct the NJ tree. IDs marked with blue dots are chickens infected with MDV. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
bottleneck effects, like those identified in the red jungle fowl (Worley et al., 2008) and blue tit (Schut et al., 2011b). Both the a1 and a2 domains were the core region of transmembrane tail that had immediate interaction with antigens, though in this study only exon 3 was found to be under a significant balancing selection. Exon 3 had a relatively higher nucleotide diversity than exon 2, which may indicate that exon 3 plays a more important role than exon 2 in antigens response or MDV response. Further study with more infected and uninfected chicken samples are needed to validate our speculation. 4.4. No specific pathogen-driven selection was observed in exon 2 and exon 3 Our analysis revealed no significant clustering difference either in exon 2 or exon 3. Compared to sequences that we obtained in this study, WL, BJY and blue-egg caipira groups were remarkably clustered together in the tree based on exon 2. The more confusing and complex clustering pattern in the tree based on exon 3 gave some credence to our supposition that exon 3 plays a more active role in responding to environmental pathogens than exon 2. The selection pressure from comprehensive and consistent pathogens was more rational to challenge the elucidation, and it was coincided with the study of promiscuous peptide bonding of MHC class I (Koch et al., 2007). The inconspicuous differences in phylogenetic
trees of exon 2 and exon 3 between the infected and uninfected chickens may indicate promiscuous pathogens effects or genetic drift, but it is difficult to make any definitive conclusion based on the evidence gathered in this study. In either exon 2 or 3, blue-egg caipira group was clustered with haplotype B21, which may proclaim that blue-egg caipira had stronger MDV-resistance than other breeds tested in this study. However, the fact the four groups AA, A03, E1, Huiyang were clustered together with B6 and B12 might demonstrate a neutral resistance to MDV, which can be validated by the results of our MDV challenge wherein MD lesion morbidity was higher than B21 and lower than B19. Ultimately, such a resistance is likely due to long-term adaptation and efficacy of the chicken’s unique pathogen environment (Jeffery and Bangham, 2000).
5. Conclusion The incidence of lesions centrally appeared between 21 and 35 dpc, and that indigenous chickens did not necessarily fare better than commercial broilers in terms of disease resistance. The challenge results and subsequent phylogenetic analysis validated the neutral behavior of all four chicken groups to MDV resistance. In total, there were 12 variable sites for exon 2 and 11 sites for exon 3, which exclusively appeared in infected chickens. Further
Y. Wang et al. / Infection, Genetics and Evolution 21 (2014) 103–109
analysis led us to speculate that exon 3 may be more crucial than exon 2 in disease resistance, and moreover, not only MDV but also promiscuous pathogens might exert selection pressure on exon 3 of MHC class I. Acknowledgement We would like to thank Prof. Dingming Shu at the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences for his assistance in chicken MDV challenge experiment. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.meegid.2013.10.020. References Abdul-Careem, M., Hunter, B., Lee, L., Fairbrother, J., Haghighi, H., Read, L., Parvizi, P., Heidari, M., Sharif, S., 2008. Host responses in the bursa of fabricius of chickens infected with virulent Marek’s disease virus. Virology 379, 256–265. Abdul-Careem, M.F., Javaheri-Vayeghan, A., Shanmuganathan, S., Haghighi, H.R., Read, L.R., Haq, K., Hunter, D.B., Schat, K.A., Heidari, M., Sharif, S., 2009. Establishment of an aerosol-based Marek’s disease virus infection model. Avian Dis. 53, 387–391. Borghans, J.A.M., Beltman, J.B., Boer, R.J., 2004. MHC polymorphism under host– pathogen coevolution. Immunogenetics 55, 732–739. Bumstead, N., Kaufman, J., 2004. Genetic resistance to Marek’s disease. In: Davison, F., Nair, V. (Eds.), Marek’s Disease, An Evolving Problem. Elsevier Ltd., London, pp. 112–125. Chaves, L.D., Faile, G.M., Krueth, S.B., Hendrickson, J.A., Reed, K.M., 2010. Haplotype variation, recombination, and gene conversion within the turkey MHC-B locus. Immunogenetics 62, 465–477. Cloutier, A., Mills, J.A., Baker, A.J., 2011. Characterization and locus-specific typing of MHC class I genes in the red-billed gull (Larus scopulinus) provides evidence for major, minor, and nonclassical loci. Immunogenetics 63, 377–394. Dalgaard, T., Boving, M.K., Handberg, K., Jensen, K.H., Norup, L.R., Juul-Madsen, H.R., 2009. MHC expression on spleen lymphocyte subsets in genetically resistant and susceptible chickens infected with Marek’s disease virus. Viral Immunol. 22, 321–327. Dalgaard, T., Højsgaard, S., Skjødt, K., Juul-Madsen, H.R., 2003. Differences in chicken major histocompatibility complex (MHC) class Ia gene expression between Marek’s disease-resistant and -susceptible MHC haplotypes. Acand. J. Immunol. 57, 135–143. Eimes, J.A., Bollmer, J.L., Dunn, P.O., Whittingham, L.A., Wimpee, C., 2010. MHC class II diversity and balancing selection in greater prairie-chickens. Genetica 138, 265–271. Emmerson, D., 1997. Commercial approaches to genetic selection for growth and feed conversion in domestic poultry. Poultry Sci. 76, 1121–1125. Fulton, J.E., Juul-Madsen, H.R., Ashwell, C.M., McCarron, A.M., Arthur, J.A., O’Sullivan, N.P., Taylor, R.L., 2006. Molecular genotype identification of the Gallus gallus major histocompatibility complex. Immunogenetics 58, 407–421. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser., 95–98. Hedrick, P.W., 1998. Balancing selection and MHC. Genetica 104, 207–214. Hepkema, B., Hensen, E., Blankert, J., Zijpp, A., Albers, G., Tilanus, M., Egberts, E., 1993. Mapping of susceptibility to Marek’s disease within the major histocompatibility (B) complex by refined typing of white leghorn chickens. Anim. Genet. 24, 283–287.
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Hunt, H., Lupiani, B., Miller, M., Gimeno, I., Lee, L., Parcells, M., 2001. Marek’s disease virus down-regulates surface expression of MHC (B complex) class I (BF) glycoproteins during active but not latent infection of chicken cells. Virology 282, 198–205. Jeffery, K.J.M., Bangham, C.R.M., 2000. Do infectious diseases drive MHC diversity? Microbes Infect. 2, 1335–1341. Jin, Y.C., Wei, P., Wei, X.X., Zhao, Z.Y., Li, Y., 2010. Rapid detection of BF haplotypes by a semi-nested polymerase chain reaction, which causes resistance/ susceptibility to Marek’s disease in chicken. Scand. J. Immunol. 72, 94–97. Kim, D., Lillehoj, H., Hong, Y., Park, D., Lamont, S., Han, J., Lillehoj, E., 2008. Immunerelated gene expression in two B-complex disparate genetically inbred fayoumi chicken lines following Eimeria maxima infection. Poultry Sci. 87, 433–443. Koch, M., Camp, S., Collen, T., Avila, D., Salomonsen, J., Wallny, H.J., van Hateren, A., Hunt, L., Jacob, J.P., Johnston, F., 2007. Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding. Immunity 27, 885– 899. Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452. Liu, L., Wu, C., Wen, J., Chen, J., Zheng, M., Zhao, G., 2009. Association of SNPs in exon 2 of the MHC BF gene with immune traits in two distinct chicken populations: Chinese Beijing-You and white leghorn. Acta Agr. Scand. A–AN 59, 4–11. Niikura, M., Kim, T., Hunt, H.D., Burnside, J., Morgan, R.W., Dodgson, J.B., Cheng, H.H., 2007. Marek’s disease virus up-regulates major histocompatibility complex class II cell surface expression in infected cells. Virology 359, 212–219. Owen, J.P., Delany, M.E., Mullens, B.A., 2008. MHC haplotype involvement in avian resistance to an ectoparasite. Immunogenetics 60, 621–631. Piertney, S., Oliver, M., 2005. The evolutionary ecology of the major histocompatibility complex. Heredity 96, 7–21. Prugnolle, F., Manica, A., Charpentier, M., Guégan, J.F., Guernier, V., Balloux, F., 2005. Pathogen-driven selection and worldwide HLA class I diversity. Curr. Biol. 15, 1022–1027. Richardson, D., Westerdahl, H., 2003. MHC diversity in two Acrocephalus species: the outbred great reed warbler and the inbred Seychelles warbler. Mol. Ecol. 12, 3523–3529. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Schou, T., Labouriau, R., Permin, A., Christensen, J., Sørensen, P., Cu, H., Nguyen, V., Juul-Madsen, H., 2010. MHC haplotype and susceptibility to experimental infections (SalmonellaEnteritidis, Pasteurella multocida or Ascaridia galli) in a commercial and an indigenous chicken breed. Vet. Immunol. Immunopathol. 135, 52–63. Schut, E., Aguilar, J.R., Merino, S., Magrath, M.J.L., Komdeur, J., Westerdahl, H., 2011. Characterization of MHC-I in the blue tit (Cyanistes caeruleus) reveals low levels of genetic diversity and trans-population evolution across European populations. Immunogenetics 63, 531–542. Sharma, J., Stone, H., 1972. Genetic resistance to Marek’s disease. Delineation of the response of genetically resistant chickens to Marek’s disease virus infection. Avian Dis., 894–906. Shiina, T., Briles, W.E., Goto, R.M., Hosomichi, K., Yanagiya, K., Shimizu, S., Inoko, H., Miller, M.M., 2007. Extended gene map reveals tripartite motif, C-type lectin, and Ig superfamily type genes within a subregion of the chicken MHC-B affecting infectious disease. J. Immunol. 178, 7162–7172. Silva, M.C., Edwards, S.V., 2009. Structure and evolution of a new avian MHC class II B gene in a sub-Antarctic seabird, the thin-billed prion (Procellariiformes: Pachyptila belcheri). J. Mol. Evol. 68, 279–291. Spurgin, L.G., Richardson, D.S., 2010. How pathogens drive genetic diversity: MHC, mechanisms and misunderstandings. P. Roy. Soc. B Biol. Sci. 277, 979–988. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Westerdahl, H., Wittzell, H., von Schantz, T., 2000. MHC diversity in two passerine birds: no evidence for a minimal essential MHC. Immunogenetics 52, 92–100. Worley, K., Gillingham, M., Jensen, P., Kennedy, L., Pizzari, T., Kaufman, J., Richardson, D., 2008. Single locus typing of MHC class I and class II B loci in a population of red jungle fowl. Immunogenetics 60, 233–247.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Sequence variations of the MHC class I gene exon 2 and exon 3 between infected and uninfected chickens challenged with Marek’s disease virus Ye Wang a,1, Mohan Qiu b,1, Jiandong Yang c, Xiaoling Zhao a, Yan Wang a, Qing Zhu a, Yiping Liu a,⇑ a
Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Chengdu Campus, Chengdu, Sichuan 611130, China Sichuan Animal Science Academy, Chengdu, Sichuan 610066, China c College of Animal Science and Technology, Sichuan Agricultural University, Ya’an, Sichuan 625014, China b
a r t i c l e
i n f o
Article history: Received 23 May 2013 Received in revised form 21 October 2013 Accepted 24 October 2013 Available online 4 November 2013 Keywords: Chicken MDV challenge MHC class I exon 2 Exon 3 Evolutionary differences
a b s t r a c t The major histocompatibility complex (MHC) among chickens has been well established as being associated with disease resistance and pathogens infection, but the genetic differences in MHC between chickens susceptible to certain infections and those chickens that remain uninfected have not been sufficiently determined. In this study, we sought the genetic basis that may underlie differences in susceptibility to infection among chickens by challenging four groups of broilers with Marek’s disease virus (MDV). Over the course of the experiment, lesions began to appear between 21 and 35 days post challenge (dpc), and commercial broilers were not necessarily better than indigenous chickens in terms of disease resistance. The four groups showed neutral resistance to MDV infection validated by challenge results and evolutionary analysis of exons 2 and 3 of the MHC class I region. Several variable sites in exon 2 and exon 3 were exclusively appeared in infected chickens. Exon 3 was likely more crucial than exon 2 in disease resistance. Our observations offered a support for a potential association between promiscuous pathogens and conspicuous genetic diversity in the MHC class I region. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The chicken major histocompatibility complex (MHC) is recognized as the most polymorphic coding region known in the vertebrate genome, with strong associations to both disease resistance and pathogen infection (Chaves et al., 2010; Eimes et al., 2010; Fulton et al., 2006). The MHC itself is comprised of two genes (MHC class I and class II) and encodes membrane glycoprotein receptors that present foreign peptides for T cells – likely a critical mechanism in scheduling immune responses (Silva and Edwards, 2009). Though intriguing for the sheer complexity of the MHC’s polymorphic nature, gaining a clearer understanding of the precise function of the MHC has broad implications. Marek’s disease (MD), for example, is a highly contagious T-cell lymphoma caused by Marek’s disease virus (MDV) that causes significant economic losses in the production of poultry. A prior study on MDV indicated that the genes associated with the B complex involved in resistance to MD are localized within the B–F/B–L region (Hepkema et al., 1993), but a more recent and clearer picture indicated that MDV is capable of down-regulating ⇑ Corresponding author. Address: Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Chengdu Campus, 211# Huimin Road, Wenjiang, Sichuan 611130, China. Tel./fax: +86 28 86290987. E-mail address: [email protected] (Y. Liu). 1 These authors contributed equally to this work, and shall share the first author. 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.10.020
the surface expression of the MHC class I gene in the immediate early phase, a process completed by the early phase of viral gene expression (Hunt et al., 2001). Similarly, recent studies validated the differences of class I expression between both MD-resistant and -susceptible chickens (Dalgaard et al., 2003, 2009). The currently verified ‘‘minimal essential MHC’’ hypothesis suggests that in chickens, the abundant class I alleles result in either the resistance or susceptibility to specific pathogens (Kim et al., 2008; Owen et al., 2008). The Class I molecule itself consists of one alpha chain and b2-microglobuin, of which, the alpha chain is the transmembrane tail which is composed by domains a1, a2, a3 (Jin et al., 2010). Indeed, a1 and a2 are the more important domains that assist in the formation of the residues present in antigen bonding region, which are encoded by the exons 2 and 3 of class I, respectively (Cloutier et al., 2011). Accordingly, the exon 2 and 3 regions would suffer from most pathogen-driven forces and selection pressures, which may explain the previously observed accumulation of diversity and polymorphisms as well as the remarkable complexity of the chicken MHC (Bumstead and Kaufman, 2004; Piertney and Oliver, 2005). Among chickens, serological studies have identified MD-resistant and -susceptible haplotypes, such as B21 and B19, but little has been done to explain the actual molecular differences of key regions of the MHC class I between chickens that are either MD-resistant or -susceptible. In this study, we opted to examine the underlying genetic basis that may help explain resistance or susceptibility to MD by
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challenging four groups of chickens with a very virulent (vv) MDV before sequencing exons 2 and 3 of the MHC class I gene in order to (a) screen the sequence variations across four chicken groups, and (b) to investigate whether the resulting infected or uninfected chickens were under diverse selection pressures or had any significant differences between them. 2. Materials and methods
Dalian, China). A standard HotStart PCR process was used for PCR amplification, which is comprised of an initial denaturation cycle of 5 min at 94 °C, 35 cycles at 94 °C for 45 s, annealing at 62 °C (for exon 2) and 58.8 °C (for exon 3) for 45 s, and 72 °C for 50 s, followed by a final extension cycle at 72 °C for 8 min, ending with an incubation at 4 °C. PCR products were checked on 1% agarose gel, following which all PCR products were purified on spin columns and sequenced by Invitrogen Corporation Shanghai (Shanghai, China).
2.1. Chickens 2.5. Data analysis All procedures used in this study were in accordance with the guide for the care and use of laboratory animals. In brief, we collected 193 25-day old broiler chickens for challenging with MDV from four groups: 49 Huiyang chickens – famous Chinese indigenous chickens known for their meat quality; 44 AA broilers from commercial lines; and 50 A03 chickens and 50 E1 chickens from specialized lines, which were bred and maintained at the Institute of Animal Science, Guangdong Academy of Agricultural Sciences. Prior to challenge with MDV, all chickens were tested to ensure they were specific-pathogen-free (SPF), i.e., free from any antibodies to MDV, Newcastle disease virus, reovirus, chicken anaemia virus (CAV), infectious bursal disease virus (IBDV). All chickens were also kept in isolators drinking ad libitum with commercial food, to maintain integrity of the experiment. 2.2. MDV challenge and postmortem Blood samples were taken from each individual prior to the MDV challenge, and chickens were challenged by intra-abdominal injection with the very virulent virus MDV J-1 strain, which is a virulent reference strain in China (provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences), at a dosage of 1000 PFU (plaque forming unit) per chicken in 0.2 ml of diluent. Clinical symptoms and mortality were observed daily for each individual. At 21 days post challenge (dpc), we began euthanizing chickens, 10 birds per group each week, until at 41 dpc all remaining birds were sacrificed for postmortem analysis. In the interim, we conducted executed postmortem examination for chickens either dying or which were euthanized to examine gross MD tumor lesions. 2.3. Genomic DNA extraction and primer design Following MDV challenge, chickens from the four groups (Huiyang, AA broilers, A03 and E1 lines) that had either became infected or remained uninfected were randomly chosen and placed into two groups for sequencing (Table S1) of either exon 2 (44 individuals) or exon 3 (47 individuals). Genomic DNA was extracted from whole blood by the standard phenol/chloroform method. Two primer pairs (primer pair 1, F: 50 -ccgcccgtaaccccaccc-30 /R: 50 -agcccatcccacacccacgg-30 ; primer pair 2, F: 50 -cgcggctccgtgggtgtgg-30 / R: 50 -cgcggctccgtgggtgtgg-30 ) were designed according to the chicken reference sequence (GenBank accession no. M31012) to amplify exons 2 (396 bp fragment) and 3 (501 bp fragment), respectively. Primer pairs were synthesized by Invitrogen Corporation Shanghai (Shanghai, China). 2.4. PCR amplification and sequencing PCR amplification was performed in a 50 lL reaction mixture containing 100 ng of DNA, 10 mmol/L Tris–HCl (pH 8.3), 2.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mM of each dNTPs, 10 pmol/L of each primer, and 1 unit of Taq polymerase (Takara,
In total, 44 individuals were sequenced for exon 2 and 47 individuals were sequenced for exon 3. Sequences were edited and assembled using BioEdit 7.1.11 (Hall, 1999). For comprehensive analysis, 21 exon 2 sequences from GenBank were included, which were composed of 5 White leghorn chickens (WL) (accession numbers EU747296.1, EU747294.1, EU747292.1, EU747293.1, EU747295.1), 8 Beijing You chickens (BJU) (accession numbers EU755019.1, EU755017.1, EU755015.1, EU755013.1, EU755011.1, EU755020.1, EU755018.1, EU755016.1), 8 blue-egg chickens (accession numbers AY489145.1, AY489147.1, AY489149.1, AY489151.1, AY489155.1, AY489157.1, AY489159.1, AY489153.1). Similarly, 15 exon 3 sequences from GenBank were included for the analysis, which were composed of 4 HH line chickens (accession numbers AY725441.1, AY725439.1, AY725445.1, AY725447.1), 3 LL line chickens (accession numbers AY725453.1, AY725451.1, AY725449.1), 8 blue-egg chickens (accession numbers AY489145.1, AY489147.1, AY489149.1, AY489151.1, AY489155.1, AY489157.1, AY489159.1, AY489153.1). 13 sequences from GenBank were used in the analysis of both exon 2 and exon 3, which were composed of 7 B21 haplotype sequences (accession numbers S78682, AY234769, AF231499, AF231503, AF231502, AF231500 and AF231501), 2 B19 haplotype sequences (accession numbers Z54317 and Z54318), 1 B2 haplotype sequence (accession number AB426141), 1 B6 haplotype sequence (accession number Z54325), 1 B12 haplotype sequence (accession number Z54326) and sequence M31012.1. MEGA 5.1 (Tamura et al., 2011) was used to perform multiple sequence alignment and the generation of amino acid sequences. DnaSP 4.50.3 (Librado and Rozas, 2009) was used to analyze the number of haplotypes (h), haplotype diversity (Hd), nucleotide diversity (p), the average number of nucleotide differences (K). The numbers of synonymous (dS) and non- synonymous (dN) substitutions per site were also estimated. Phylogenetic trees were reconstructed using the neighbor joining (NJ) method (Saitou and Nei, 1987), and the MHC haplotypes B21 and B19 were taken into consideration to assist in judging the relative resistance and susceptibility of the four groups of chickens. 3. Results 3.1. Incidence of Marek’s disease after challenge Excluding four chickens that died during the course of the study, either without artificial interference or from MDV infection, the remaining chickens were executed on a precise schedule as stated earlier, leaving 19.2% of total chickens (37/193) significantly infected with MDV (Table 1). The incidence of MDV infection during each period of time for each breed is shown in Table 1. Overall, A03 chickens had the highest morbidity ratio (28.6%) while the AA chicken had the lowest ratio (11.6%), with the susceptibility order of the four lines as A03, E1, Huiyang, and AA. The morbidity of MD mainly occurred between 21 dpc and 35 dpc. No infected chicken was found at 42 dpc or even at 35 dpc in the E1 population.
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Y. Wang et al. / Infection, Genetics and Evolution 21 (2014) 103–109 Table 1 Morbidity and mortality rates at various time intervals following MDV challenge among four chicken lines. Chicken lines
N
AA A03 E1 Huiyang
43 49 48 49
Tumor incidence at different intervals 21 dpc
28 dpc
35 dpc
42 dpc
20% 70% 60% 50%
10% 40% 40% 30%
20% (2/10) 20% (2/10) 0% (0/10) 10% (1/10)
0% 0% 0% 0%
(2/10) (7/10) (6/10) (5/10)
(1/10) (4/10) (4/10) (3/10)
(0/13) (0/18) (0/17) (0/18)
Morbidity rate
Mortality rate
11.6% 28.6% 22.9% 20.4%
– 7.1% (1/14) 9.1% (1/11) 10% (1/10)
(5/43) (14/49) (11/48) (10/49)
N is the valid number of chickens that died either due to MDV challenge or human interference. ‘‘–’’ denotes no chicken died during the course of challenge.
3.2. Variation and high levels of diversity of exon 2
whole population were 0.04002 and 10.924, respectively. Among the four groups, A03 had the highest nucleotide diversity (0.03914) and nucleotide differences (10.684), E1 was next to A03 (p = 0.03810; K = 10.400), and Huiyang chickens had the lowest nucleotide diversity (0.02821) and nucleotide differences (7.700). Theta values were 0.02911, 0.03983, 0.03627 and 0.02989 for AA, A03, E1 and Huiyang, respectively, yielding an average value of 0.03815. The Tajima’s D test had a positive value of 0.23314 in E1 population, while the other three populations were negative (AA, 0.10613; A03, 0.06954; Huiyang, 0.41429) (Table 2).
A total of 77 sequences (including 33 reported sequences; with a truncated length of 265 bp) were analyzed for exon 2. There was no length variation except for a base insertion at the 100th position for individual C195 (Table S2). Sixty-nine variable sites were found, yielding a mean nucleotide mutation rate of 26.04% (69/265). Among these variable sites, 16 were singleton variants, 53 were parsimony informative sites. Interestingly, variations at sites 12 (C > A), 195 (C > G), 205 (A > C), and 237 (A > T) appeared exclusively to infected chickens. Analysis of exon 2 showed high levels of haplotype diversity (H) among the four groups, with values of 0.985 for AA, 0.994 for A03, 1.000 for E1, and 1.000 for Huiyang chicken (Table 2). High levels of haplotype diversity indicated abundant nucleotide diversity, in this case AA had the highest p (0.04477) and K values (11.818), whereas A03 had the lowest values for both p (0.02924) and K (7.719) among the four populations. Theta-W and Tajima’s D were also calculated for each of the four groups to further clarify these initial observations. The polymorphism index of Theta values were 0.04641, 0.03035, 0.03080 and 0.04040 for AA, A03, E1 and Huiyang, respectively. The Huiyang chicken and E1 chicken had a positive Tajima’s D value, while populations AA (0.16084) and A03 (0.14386) had a negative value (Table 2). There was no parsimony informative site in the Huiyang chickens, perhaps due to its modest sample size.
3.4. Different selection pressures between infected and uninfected chickens As the antigen bonding region, the a1 and a2 domains encoded by exons 2 and 3 might have undergone the strongest pathogen selection. We calculated the values of dN/dS and executed a codon-based Z test to determine whether there was a selection in those two regions (Table 3). The peptide binding region (PBR) was inferred and superimposed on sequences using previously published data from a great reed warbler (Richardson and Westerdahl, 2003; Westerdahl et al., 2000). We found no significant selection in exon 2, while the PBR had a higher dN/dS value (2.017, P = 0.97) than non-PBR (0.979, P = 0.48). The uninfected groups generally had higher dN/dS values than infected ones. However, analysis did show that the PBR of exon 3 was under significant balancing selection (4.356, P = 0.02) while the non-PBR of exon 3 were under an unapparent purifying selection (0.501, P = 0.09). Interestingly, in the PBR of uninfected chickens, the dN/dS value was significant (7.253, P = 0.01) and much higher than that found among the infected chickens (2.400, P = 0.05), while this was not found in either the non-PBR or the other remaining sites. Compared to exon 2, exon 3 also had a lower dN/dS value (PBR: 1.123, P = 0.35; nonPBR: 0.531, P = 0.08), suggesting that exon 2 might suffer balancing selection while exon 3 was under purifying selection.
3.3. Variation and high levels of diversity of exon 3 Analysis of 75 sequences (including 28 reported sequences) of exon 3 yielded a total of 55 variable sites along the 273 bp fragment (Table S3). Of the 55 variable sites, six were singleton variable sites and 49 were parsimony informative sites. Intriguingly, variable sites 11, 178, 199 were found only in infected group. Exon 3 showed a markedly high level of haplotype diversity, with an average of haplotype diversity (H) 0.998 (Table 2). The overall nucleotide diversity and nucleotide differences of the
Table 2 Nucleotide diversity of MHC class I exon 2 and exon 3 among four chicken populations. Chicken lines Exon 2 AA A03 E1 Huiyang Total Exon 3 AA A03 E1 Huiyang Total
N
No. of polymorphic sites
No. of parsimony informative sites
K
p
Theta (per site) from Eta
Tajima’s D
Significance
12 19 10 3 44
34 26 23 14 38
25 25 18 0 31
11.818 7.719 8.422 10.000 9.795
0.04477 ± 0.00746 0.02924 ± 0.00514 0.03190 ± 0.00762 0.03788 ± 0.01045 0.03710 ± 0.00368
0.04641 0.03035 0.03080 0.04040 0.04006
0.16084 0.14386 0.17025 0.25784
P > 0.10 P > 0.10 P > 0.10 – P > 0.10
12 19 11 5 47
21 31 27 16 39
14 23 20 5 31
7.758 10.684 10.400 7.700 10.924
0.02842 ± 0.00259 0.03914 ± 0.00303 0.03810 ± 0.00405 0.02821 ± 0.00790 0.04002 ± 0.00204
0.02911 0.03983 0.03627 0.02989 0.03815
0.10613 0.06954 0.23314 0.41429 0.16933
P > 0.10 P > 0.10 P > 0.10 P > 0.10 P > 0.10
K is average number of nucleotide difference; p is average number of nucleotide diversity.
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Table 3 Estimates of average codon-based evolutionary divergence and positive selection in chickens challenged by MDV. Region
Infection status
PBR
Infected Uninfected Overall Infected Uninfected Overall Infected Uninfected Overall
Non-PBR
All sites
Exon 2
Exon 3
dN (±SE)
dS (±SE)
dN/dS
p
dN (±SE)
dS (±SE)
dN/dS
p
0.0650 ± 0.0196 0.0887 ± 0.0323 0.0823 ± 0.0030 0.0283 ± 0.0093 0.0305 ± 0.0094 0.0297 ± 0.0088 0.0361 ± 0.0009 0.0424 ± 0.0103 0.0412 ± 0.0098
0.0415 ± 0.0428 0.0408 ± 0.0402 0.0407 ± 0.0388 0.0294 ± 0.0137 0.0203 ± 0.0100 0.0303 ± 0.0136 0.0373 ± 0.0125 0.0358 ± 0.0130 0.0367 ± 0.0125
1.568 2.177 2.017 0.963 1.503 0.979 0.968 1.187 1.123
0.28 0.14 0.16 0.46 0.21 0.48 0.46 0.28 0.35
0.0773 ± 0.0322 0.0683 ± 0.0275 0.0706 ± 0.0283 0.0302 ± 0.01172 0.0283 ± 0.0105 0.0270 ± 0.0101 0.0360 ± 0.0108 0.0284 ± 0.0078 0.0284 ± 0.0079
0.0274 ± 0.0265 0.0094 ± 0.0093 0.0162 ± 0.0162 0.0569 ± 0.0205 0.0531 ± 0.0183 0.0539 ± 0.0200 0.0552 ± 0.0177 0.0540 ± 0.0174 0.0535 ± 0.0017
2.400 7.253 4.356 0.532 0.533 0.501 0.651 0.525 0.531
0.05 0.01 0.02 0.09 0.09 0.09 0.15 0.08 0.08
Note: analyses of the numbers of synonymous (dS) and non-synonymous (dN) substitutions per site were conducted using the Nei–Gojobori method in MEGA 5.1. Standard error estimates are obtained by a bootstrap procedure (2000 replicates). All positions containing alignment gaps and missing data were eliminated. p Values were calculated by the codon-based Z test with the alternative hypothesis of dN > dS of dN < dS. PBR, peptide binding region; Non-PBR, region beyond the peptide binding region.
3.5. Phylogenetic trees of exon 2 and exon 3 The unrooted NJ tree for exon 2 among the four groups, along with the aforementioned sequences from GenBank, had a complex clustering pattern (Fig. 1). Chickens from the four groups were intermixed together with a blear divergence. There was no significant clustering between infected individuals and uninfected ones. This clustering pattern hardly changed when haplotypes B6 and B12 were included in the analysis. B2 was clustered with one blue-egg chicken in either exon 2 or exon 3. Likewise, WL, BJY and blue-egg caipira groups were apparently clustered together, most blue-egg caipiras were clustered together with haplotype B21, and WL and BJY were mixed with each other. Similarly, for exon 3, no significant clustering differences were found between infected individuals and uninfected chickens (Fig. 2). The complicated phylogenetic tree of exon 3 showed that the seven lines were permeated with each other. 4. Discussion A variety of studies have demonstrated that the chicken MHC gene was extensively associated with pathogens infection like avian influenza (AI), Newcastle disease (ND), Marek’s disease virus (MDV) (Abdul-Careem et al., 2008; Liu et al., 2009; Niikura et al., 2007; Schou et al., 2010; Shiina et al., 2007). This finding is consistent with the basic premises of an ongoing ‘‘evolutionary arms race’’ between the large numbers of pathogens in a given environment and the MHC gene of given hosts gave rise to the observed high genetic diversity of the MHC, which was presumed to be maintained via balancing selection (Borghans et al., 2004). In this study, we conducted a MDV challenge on four chicken groups (AA, A03, E1, Huiyang) followed by genetic sequencing of the MHC gene. We further used available data from WL, BJY, blueegg caipira, HH and LL chicken lines to construct a sequence profile of exons 2 and 3 of the MHC class I gene in order to gain a more complete picture of sequence diversity, balancing selection and phylogenetic tree of the chickens challenged with MDV. While the low MDV infection ratio and limited experimental capacity prevent more practical samples apply to our study, further investigation would be helpful to solidify our current observation. 4.1. Infection characteristic of MDV challenge MDV challenge resulted in the first death of a chicken with MD lesions at 20 dpc, with the incidence of lesions centrally appearing sometime between 21 dpc and 35 dpc, which were consistent with previous findings (Abdul-Careem et al., 2009; Sharma and Stone, 1972). In general, the morbidity of MD lesions was 19.2% (37/
193), though this was relatively lower than the rate found among the chickens with B19 haplotypes but higher than the chickens with B21 haplotypes (Dalgaard et al., 2003). Though in no way a conclusive finding, this result offers some intriguing circumstantial evidence that supports our findings on phylogeny clustering. One cautionary note is worth mentioning, these results could also support an inference that indigenous chicken breeds may not possessed better behavior to disease resistance than commercial chicken breeds, as the AA broilers had a striking lower morbidity and mortality than our local breed Huiyang chicken. This could instead be explained by gene exchange and heterosis, which has improved the disease resistance of chickens without fundamentally altering the composition of the MHC. 4.2. Polymorphisms of exons 2 and 3 of the MHC class I indicated promiscuous peptide bonding Many variable sites were found both in exon 2 (18.94%) and exon 3 (16.12%) with an uneven distribution, which were retrenched to 202–249 sites and 102–199 sites, respectively. Transferred into amino acid sequences, over half of the peptide bonding sites (PBS) were variable for exon 2 (58.8%), with slightly less in exon 3 (46.7%). Those highly variable regions may have led to a high resolution to antigens and help in recognizing novel pathogens to ensure rapid adaptation to environmental changes (Schut et al., 2011a). Moreover, this inference has been verified in human HLA class I gene, in which positive correlations were identified between HLA genetic diversity and pathogen richness (Prugnolle et al., 2005). Similarly, host-pathogen co-evolution has been purported to be the key factor of MHC polymorphism (Borghans et al., 2004). Among the four groups (AA, A03, E1, Huiyang), AA was the most variable group while A03 was the least diverse group concerning the exon 2 variation. As for exon 3, A03 was the most variable group, and Huiyang was the least varied. This is largely consistent with the realities of the modern poultry industry. To cater to the special market requirements, multiple chicken lines were utilized to construct the commercial chicken production system, which resulted in a potentially unintended consequence of leading to complex genetic variation among commercial chickens (Emmerson, 1997). This may explain the pattern for high polymorphisms of both the AA and A03 commercial lines. The diverse pattern for exon 2 and exon 3 suggests that the two exons might perform distinctly different functions during the antigen bonding process, though the actual physical distance between exons 2 and 3 was relatively compact. The binding groove of MHC class I, however, has an unusually large central cavity, which confers substantial conformational flexibility to the crucial residue Arg 9, which then in turn allows the remodeling of key peptide-binding sites (Spurgin
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Fig. 1. Phylogenetic tree of exon 2 of MHC class I sequences. 77 sequences (including 33 reported sequences) were used to reconstruct the NJ tree. IDs marked with blue dots are chickens infected with MDV. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and Richardson, 2010). Taken together, the aforementioned evidence and the high levels of polymorphism do offer some insights that support a potential association between promiscuous pathogens and conspicuous genetic diversity. 4.3. Balance selection was acted on a3 domain Given our current knowledge on MHC and the role pathogens play in evolution, it was reasonable to expect that pathogen driven selections caused by MDV occurred during chickens’ evolution history (Spurgin and Richardson, 2010), and that in turn balancing selection acted as the major mechanism to maintain variations of MHC genes (Hedrick, 1998). In this study, we calculated dN/dS value of exon 2 and exon 3 between both infected and uninfected groups using the PBR and non-PBR regions to identify whether the a1 and a2 domains were under balancing selection or not, and to check whether the differential selection pressure between infected and uninfected chickens exists. Balancing selection was significantly
found in the PBR of exon 3 and PBR of exon 2 without predominance (Table 3). Of all the PBRs, 10/17 of exon 2, and 7/15 of exon 3 were variable, which suggested that only sites with an immediate association to pathogens could be placed under acute selection pressure. These results and the subsequent inference on pathogens and selection pressure are consistent with previous studies on blue tits (Cyanistes caeruleus) (Schut et al., 2011a) and red-billed gulls (Larus scopulinus) (Cloutier et al., 2011). Collectively, the above findings and the previous researches implicate a preference for balancing selection on sites that immediately contribute to antigen bonding, as opposed to other conserved sites. Rather intriguingly, nearly all uninfected groups had a higher dN/dS value than infected groups, whether in terms of PBR sites, non-PBR sites, or all sites in general. This discrepancy may be the result of intense purifying selection caused by MDV mortality. Pathogens are a key environmental factor in reducing or controlling the adaptation of animals, which would in turn induce purifying selection and play a important role in creating population
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Fig. 2. Phylogenetic tree of exon 3 of MHC class I sequences. 75 sequences (including 28 reported sequences) were used to reconstruct the NJ tree. IDs marked with blue dots are chickens infected with MDV. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
bottleneck effects, like those identified in the red jungle fowl (Worley et al., 2008) and blue tit (Schut et al., 2011b). Both the a1 and a2 domains were the core region of transmembrane tail that had immediate interaction with antigens, though in this study only exon 3 was found to be under a significant balancing selection. Exon 3 had a relatively higher nucleotide diversity than exon 2, which may indicate that exon 3 plays a more important role than exon 2 in antigens response or MDV response. Further study with more infected and uninfected chicken samples are needed to validate our speculation. 4.4. No specific pathogen-driven selection was observed in exon 2 and exon 3 Our analysis revealed no significant clustering difference either in exon 2 or exon 3. Compared to sequences that we obtained in this study, WL, BJY and blue-egg caipira groups were remarkably clustered together in the tree based on exon 2. The more confusing and complex clustering pattern in the tree based on exon 3 gave some credence to our supposition that exon 3 plays a more active role in responding to environmental pathogens than exon 2. The selection pressure from comprehensive and consistent pathogens was more rational to challenge the elucidation, and it was coincided with the study of promiscuous peptide bonding of MHC class I (Koch et al., 2007). The inconspicuous differences in phylogenetic
trees of exon 2 and exon 3 between the infected and uninfected chickens may indicate promiscuous pathogens effects or genetic drift, but it is difficult to make any definitive conclusion based on the evidence gathered in this study. In either exon 2 or 3, blue-egg caipira group was clustered with haplotype B21, which may proclaim that blue-egg caipira had stronger MDV-resistance than other breeds tested in this study. However, the fact the four groups AA, A03, E1, Huiyang were clustered together with B6 and B12 might demonstrate a neutral resistance to MDV, which can be validated by the results of our MDV challenge wherein MD lesion morbidity was higher than B21 and lower than B19. Ultimately, such a resistance is likely due to long-term adaptation and efficacy of the chicken’s unique pathogen environment (Jeffery and Bangham, 2000).
5. Conclusion The incidence of lesions centrally appeared between 21 and 35 dpc, and that indigenous chickens did not necessarily fare better than commercial broilers in terms of disease resistance. The challenge results and subsequent phylogenetic analysis validated the neutral behavior of all four chicken groups to MDV resistance. In total, there were 12 variable sites for exon 2 and 11 sites for exon 3, which exclusively appeared in infected chickens. Further
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analysis led us to speculate that exon 3 may be more crucial than exon 2 in disease resistance, and moreover, not only MDV but also promiscuous pathogens might exert selection pressure on exon 3 of MHC class I. Acknowledgement We would like to thank Prof. Dingming Shu at the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences for his assistance in chicken MDV challenge experiment. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.meegid.2013.10.020. References Abdul-Careem, M., Hunter, B., Lee, L., Fairbrother, J., Haghighi, H., Read, L., Parvizi, P., Heidari, M., Sharif, S., 2008. Host responses in the bursa of fabricius of chickens infected with virulent Marek’s disease virus. Virology 379, 256–265. Abdul-Careem, M.F., Javaheri-Vayeghan, A., Shanmuganathan, S., Haghighi, H.R., Read, L.R., Haq, K., Hunter, D.B., Schat, K.A., Heidari, M., Sharif, S., 2009. Establishment of an aerosol-based Marek’s disease virus infection model. Avian Dis. 53, 387–391. Borghans, J.A.M., Beltman, J.B., Boer, R.J., 2004. MHC polymorphism under host– pathogen coevolution. Immunogenetics 55, 732–739. Bumstead, N., Kaufman, J., 2004. Genetic resistance to Marek’s disease. In: Davison, F., Nair, V. (Eds.), Marek’s Disease, An Evolving Problem. Elsevier Ltd., London, pp. 112–125. Chaves, L.D., Faile, G.M., Krueth, S.B., Hendrickson, J.A., Reed, K.M., 2010. Haplotype variation, recombination, and gene conversion within the turkey MHC-B locus. Immunogenetics 62, 465–477. Cloutier, A., Mills, J.A., Baker, A.J., 2011. Characterization and locus-specific typing of MHC class I genes in the red-billed gull (Larus scopulinus) provides evidence for major, minor, and nonclassical loci. Immunogenetics 63, 377–394. Dalgaard, T., Boving, M.K., Handberg, K., Jensen, K.H., Norup, L.R., Juul-Madsen, H.R., 2009. MHC expression on spleen lymphocyte subsets in genetically resistant and susceptible chickens infected with Marek’s disease virus. Viral Immunol. 22, 321–327. Dalgaard, T., Højsgaard, S., Skjødt, K., Juul-Madsen, H.R., 2003. Differences in chicken major histocompatibility complex (MHC) class Ia gene expression between Marek’s disease-resistant and -susceptible MHC haplotypes. Acand. J. Immunol. 57, 135–143. Eimes, J.A., Bollmer, J.L., Dunn, P.O., Whittingham, L.A., Wimpee, C., 2010. MHC class II diversity and balancing selection in greater prairie-chickens. Genetica 138, 265–271. Emmerson, D., 1997. Commercial approaches to genetic selection for growth and feed conversion in domestic poultry. Poultry Sci. 76, 1121–1125. Fulton, J.E., Juul-Madsen, H.R., Ashwell, C.M., McCarron, A.M., Arthur, J.A., O’Sullivan, N.P., Taylor, R.L., 2006. Molecular genotype identification of the Gallus gallus major histocompatibility complex. Immunogenetics 58, 407–421. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser., 95–98. Hedrick, P.W., 1998. Balancing selection and MHC. Genetica 104, 207–214. Hepkema, B., Hensen, E., Blankert, J., Zijpp, A., Albers, G., Tilanus, M., Egberts, E., 1993. Mapping of susceptibility to Marek’s disease within the major histocompatibility (B) complex by refined typing of white leghorn chickens. Anim. Genet. 24, 283–287.
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