1. Association of human leukocyte antigen class II allele and haplotypes
in chikungunya viral infection in a western Indian population
Subrat Thanapati, Aparna Hande, Rumki Das, Yogesh Gurav and Anuradha S Tripathy*
Hepatitis Group, National Institute of Virology, Pune, 130/1, Sus Road, Pashan, Pune, Maharashtra, India–411021
*Corresponding author: Tel: +091 20 26006390; Fax: +091 20 25871895; E-mail: anuradhastripathy@hotmail.com
Received 24 December 2013; revised 31 January 2014; accepted 3 February 2014
Background: Genes coding for human leukocyte antigen (HLA) class II molecules are polymorphic and have been
shown to influence susceptibility to viral diseases.
Methods: One hundred patients with acute chikungunya with and without viral load and 250 chikungunya nega-
tive controls from western India were studied for the distribution of HLA class II alleles by PCR with sequence-
specific primer (SSP) method.
Results: Frequency of DRB1*11 allele group (patients vs controls: p¼0.002, Pc¼0.036, OR¼0.21) and haplotype
DRB1*11/DQB1*03 (patients vs controls: p¼0.007, OR¼0.15) were significantly low, while haplotype DRB1*04/
DQB1*03 (patients vs controls: p¼0.042, OR¼1.94) was significantly high in the patient population. HLA
DQB1*04 allele was found only in the patient group with viral load (n¼17), suggesting possible involvement of
the same with chikungunya virus (CHIKV) replication.
Conclusions: Association of HLA-DRB1*11 and the emergence of DRB1*11/DQB1*03 & DRB1*04/DQB1*03 as resist-
ant and susceptible haplotypes towards CHIKV infection is being reported for the first time. Our results suggest that
genetic susceptibility and/or resistance to chikungunya infection may be modulated by HLA class II alleles.
Keywords: Chikungunya, HLA class II, Human leukocyte antigen, western Indian population
Introduction
Chikungunya virus (CHIKV), first isolated in 1953 in Tanzania from
infected patients, re-emerged since 2005, has caused millions of
cases throughout the Indian Ocean and Southeast Asia.1,2
Sporadic outbreaks are still on-going in several countries, inflicting
naive populations.3
India first experienced a CHIKV outbreak dur-
ing 1963–64 in Kolkata and in 1965 in Chennai.4,5
It has recently
re-emerged extensively in different parts of India since October
2005, including the state of Maharashtra.6,7
Associated low herd
immunity has made chikungunya fever a public threat and hence
it represents a major public health problem with a severe social
and economic impact.
CHIKV is a mosquito-borne virus belonging to the Alphavirus
genus of the Togaviridae family. It has a life cycle similar to
other alpha viruses and it causes sudden onset of fever, rashes,
arthritis and other accompanying symptoms.8,9
The symptoms
persist for three to seven days during the acute phase of the dis-
ease and disappear after two weeks. However, arthralgia, fatigue
and incapacitating joint pain continue for several weeks to months,
or even years.10
Some patients develop chronic arthritis syn-
drome.11
There are specific descriptions on chikungunya associated
joint disorders.12,13
Varied clinical presentations in chikungunya
infection could be attributed to the host factors and/or critical
mutations in the viral genome.14–16
The host’s innate and adaptive
immune response has been reported to play a major role in control-
ling the viral pathogenesis in CHIKV infection.17,18
There has been
an increasing interest in the understanding of the role of host gen-
etic factors in the pathogenesis of viral diseases.
Human leukocyte antigens (HLAs) are encoded by the most
polymorphic genes that present antigens to CD8+ cytotoxic and
CD4+ helper Tcells.19
HLA class II molecules have been crucial for
developing an adequate immune response. Polymorphism in HLA
class II molecules giving rise to amino acid substitutions, deter-
mines the antigenic specificities of host immune responses
against infection.20,21
HLA class II alleles have been reported to
be responsible for the selection of viral epitopes for presentation
to CD4+ T cells that lead to an efficient immune response against
the virus.22
In a study of 21 patients from Reunion Island with
chronic chikungunya, HLA-DRB1*01 and DRB1*04 alleles were
frequently found among the patients who developed rheumatoid
arthritis after the infection, indicating the probable involvement of
HLA class II gene in chikungunya infection.23
However, details
regarding the relevance of these alleles with chikungunya virus
associated clinical presentations were not indicated in the
# The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved.
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ORIGINALARTICLE
Trans R Soc Trop Med Hyg
doi:10.1093/trstmh/tru030
1
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2. study. In an attempt to understand the role of genetic factors
contributing towards the resistance and/or susceptibility to chi-
kungunya infection, we have genotyped HLA class II loci and
quantified chikungunya plasma RNA levels in a case-control
study of patients with chikungunya and racially matched healthy
controls of Maharashtra, India.
Material and methods
Study population
A population of 350 individuals was included in the current study.
The patient population (n¼100) came from different chikungunya
outbreaks in Maharashtra and the control population (n¼250)
came from various blood donation camps organized in Pune,
Maharashtra. The diagnosis of chikungunya infection was based
on the presence of IgM antibodies against virus (anti-CHIKV
IgM) as detected by ELISA. The patients were examined, their clin-
ical courses were documented and only the laboratory confirmed
chikungunya cases, i.e. positive for at least any one of the follow-
ing: CHIKV isolation in mice or tissue culture/CHIKV RNA and/or
anti-CHIKV IgM antibody positive. All the study subjects were
screened for chikungunya viral RNA by RT-PCR or anti-CHIKV
IgM/IgG antibodies by ELISA. The control group consisted of age
and sex-matched apparently healthy individuals negative for
anti-CHIKV IgM/IgG antibodies. The patients and controls
belonged to the same geographical area and same ethnic groups.
This research project was approved by the Institutional Ethical
Committee for Research on Humans as per the guidelines of the
Indian Council of Medical Research. Written consent was obtained
from all the participants involved in the study.
Serological and molecular testing
Blood samples were collected in K3 EDTA tubes and plasma was
separated. An aliquot of plasma was stored at 2708C for the
detection of viral load. All the samples were screened for
anti-CHIKV IgM and IgG antibodies, and for antibody against
Dengue virus (anti-DENV) by ELISA.24
TaqMan-based one-step real-time PCR
Plasma samples from chikungunya patients with post onset days
(POD) of illness less than 15 were screened for the quantitation of
CHIKV RNA. Briefly, RNA was extracted using QIAamp viral RNA
mini kit (Qiagen, Hilden, Germany), and viral load (copies/ml)
was determined using CHIKV E3 based primers and probe accord-
ing to the protocol described earlier.25
One-step TaqMan real time
(RT)-PCR assay was performed in a 96-well format using an ABI
PRISM 7300 sequence detection system (Applied Biosystems,
Foster City, CA, USA). Reaction conditions were: RT at 488C for
30 min, initial denaturation at 958C for 10 min, and 50 cycles of
denaturation (958C for 15 sec), followed by annealing and exten-
sion (608C for 1 min). The detection limit was 10 copies/reaction.
HLA typing
HLA typing for class II molecules was carried out in 100 chikun-
gunya patients and 250 ethnically matched healthy controls
from Maharashtra.
Genomic DNA was extracted from whole blood by Qiagen
Blood mini kit (Qiagen). Molecular typing was carried out by
PCR-sequence specific primer (SSP) provided with SSP HLA-DQ-
DR Combi Tray Kit (Olerup SSP, West Chester, PA, USA). The
DQ-DR SSP combi tray contained 5′
and 3′
primers for grouping
DQB1 *050101 to 0505, *060101 to 0639, *020102 to 0205,
*030101 to 0326, *0401 to 0404 alleles, 5′
and 3′
primers for
grouping DRB1*010101 to 1003, DRB3*01010201 to *0303,
DRB4*01010101 to *0108, DRB5*010101 to *0205 alleles.
Briefly, genomic DNA at a concentration of 30 ng/ml was used
for the amplification of DQB1 and DRB1 alleles using gene-
specific primers. PCR reaction mixture provided with the kit con-
sisted of 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2,
0.001% gelatine, 200 mM each type of dNTPs, 25 pmol of each
primer, 5% glycerol, cresol red 100 mg/ml, 0.4 Units of Taq DNA
polymerase(Roche, Burgess Hill, West Sussex, UK) was added.
Size of PCR amplified product ranged from 85–2505 bp. The
internal positive control primer pair amplified segments of the
human growth hormone (430–515 bp). PCR cycling parameters
were maintained at: 1 cycle at 948C for 2 min of denaturation, 10
cycles at 948C for 10 sec of denaturation, followed by annealing
and extension at 658C for 60 sec, remaining 20 cycles of
denaturation at 948C for 10 sec, annealing at 618C for 50 sec fol-
lowed by an extension at 728C for 30 sec. The correct size of
the product was confirmed by gel electrophoresis using a 2%
agarose gel with ethidium bromide (10 mg/ml) in 0.5xTAE
(Tris-acetate EDTA) buffer. The gel was observed under ultravio-
let exposure and allele groups were confirmed based on the
presence or absence of allele specific bands along with internal
control band.
Statistical analysis
The distributions of HLA-DRB1/DQB1 allele groups were deter-
mined using Helmberg SCORE V 4.0200 T 1 KITsoftware. The allele
frequencies were calculated by direct counting and expressed as
percentage of total number of chromosomes (2n) in each group.
Haplotype frequency was estimated by PyPopwin32–0.7.0 soft-
ware. Allele and haplotype frequencies were compared between
patients and controls using x2
with Yates correction. p values
and ORs were estimated using OpenEpi (version 2.3; http://
faculty.vas-sar.edu/lowry/tabs.html). Bonferroni correction was
applied to the p values.
Results
Clinical and virological characteristics of the patients
and controls
The clinical and virological characteristics of chikungunya
patients and control individuals enrolled in the study are
depicted in Table 1. There were no significant differences in
the age and sex of the participants. All the patients’ sam-
ples were anti-DENV negative. The complete blood count/
erythrocyte sedimentation rate, C-reactive protein, rheuma-
toid factor, anti-cyclic citrullinated peptide antibodies and
anti-nuclear antibody pattern were tested in patients’ sam-
ples to establish that the arthritis was due to chikungunya
infection.
S. Thanapati et al.
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3. HLA typing
HLA DQ-DR allele frequency in chikungunya patients and controls
Only allele frequencies of .5% in either group were considered. In
the patient as well in the control groups, HLA-DRB1*15 and
DQB1*06 were found as the most common alleles (HLA-
DRB1*15: patient 28.5% [57/200] vs control 32.2% [161/500]
and DQB1*06: patient 32.5% [65/200] vs control 35.8% [179/
500]). HLA-DRB1*01 allele was found at a relatively higher
frequency in the patient group 5.5% (11/200) compared to the
control group 2.6% (13/500), whereas DRB1*11 allele was found
at a relatively lower frequency in the patient group 2% (4/200)
compared to the control group 8.6% (43/500). Though not signifi-
cant, the frequency of DRB1*04 was relatively higher in the patient
group 12.0% (24/200) compared to the controls group 8.8% (44/
500). Overall, at the DQ-DR locus, the frequency of DRB*11 allele
was significantly low in the chikungunya patient group 2.0%
(4/200) vs control 8.6% (43/500), OR 0.21, CI 0.076–0.61,
p¼0.002, that retained its significance even after applying
Bonferroni correction (Pc¼0.03; Table 2).
HLA DQ-DR haplotype frequencies in chikungunya patients and
controls
Only haplotype frequencies .5% in either groups were consid-
ered. HLA DRB1*15/DQB1*06 was the most common haplotype
with the same frequency (25% each [50/200] in the patient
group and [125/500] in the control group) in both study groups.
In the chikungunya patients, DRB1*11/DQB1*03 was significantly
low compared to controls whereas DRB1*04/DQB1*03 haplotype
was significantly high in patients compared to the controls.
However, both the associations were susceptible to Bonferroni
correction (Table 3).
HLA Class II alleles and viral load in patients
DQB1 and DRB1 allelic distribution in 17 chikungunya patients
with viral load (POD,15) and 33 without viral load (POD,15)
were investigated (Table 4). Only allele frequencies of .5% in
either group were considered. HLA DQB1*04 was found only in
the patient group with viral load. The frequency of allele
DQB1*02 was relatively higher in the patients with viral load
group compared to the other group (17% [6/34] vs 11% [7/66]),
while the frequencies of alleles DQB1*05, DQB1*06 were relatively
lower in the patients with viral load group compared with the
patients without viral load (21% [7/34] and 29% [10/34] vs 23%
[15/66] and 38% [25/66] respectively). The frequency of DRB1*04
Table 1. Characteristics of patients and controls
Parameters Patients Controls
Study population n¼100 n¼250
Sex ratio (M:F) 2:3 103:22
Age, years: median (range) 40 (9–82) 27 (18–47)
POD: median (range) 12 (2–93) NA
Anti-CHIKV IgM Positive (n¼92);
Negative (n¼8)
Negative
Viral load (copies/ml):
median (range)
3100 (120–670000000) NA
CHIKV: chikungunya virus; NA: not applicable; POD: post onset of
days of illness.
Table 2. Statistical analysis of human leukocyte antigen (HLA) allele associations in patient vs control group
HLA allele Patient (2n¼200)
n (%)
Control (2n¼500)
n (%)
Patient vs control
p-value OR (95% CI) Pc
DQB1*02 26 (13) 73 (14.6) NS 0.87 (0.54–1.41) NS
DQB1*03 47 (23.5) 111 (22.2) NS 1.07 (0.72–1.58) NS
DQB1*05 57 (28.5) 131 (26.2) NS 1.12 (0.77–1.61) NS
DQB1*06 65 (32.5) 179 (35.8) NS 0.86 (0.60–1.22) NS
DRB1*01 11 (5.5) 13 (2.6) NS 2.18 (0.96–4.95) NS
DRB1*03 15 (7.5) 28 (5.6) NS 1.36 (0.71–2.61) NS
DRB1*04 24 (12) 44 (8.8) NS 1.41 (0.83–2.39) NS
DRB1*07 27 (13.5) 73 (15) NS 0.88 (0.55–1.42) NS
DRB1*10 17 (8.5) 41 (8.2) NS 1.04 (0.57–1.87) NS
DRB1*11 4 (2) 43 (8.6) 0.002 0.21 (0.076–0.61) 0.036
DRB1*13 11 (5.5) 28 (5.6) NS 0.98 (0.47–2.01) NS
DRB1*14 24 (12) 39 (7.8) NS 1.61 (0.94–2.75) NS
DRB1*15 57 (28.5) 161 (32.2) NS 0.83 (0.58–1.20) NS
Human leukocyte antigen allele frequencies .5% in patient/control group is considered.
2n: total number of chromosomes; n: allele frequency; NS: not significant; Pc: Bonferroni corrected p-value.
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4. was relatively lower (6% [2/34] vs 9% [6/66]), while the frequen-
cies of alleles DRB1*13 and DRB1*15 were relatively higher in the
patients with viral load compared to patients without viral load
(12% [4/34] and 32% [11/34] vs 8% [5/66] and 27% [18/66]
respectively) (Table 4).
Discussion
Genetic factors of the patients and the response of CD4+ T cells
strongly influence the course of viral infections. In the same con-
text, even though various HLA class II alleles have been asso-
ciated as resistant/susceptible alleles in viral infections, few
studies have characterized host genetic determinants associated
with alphavirus infections. In the current study, we have assessed
the HLA class II alleles and haplotypes distributions in acute chi-
kungunya patients with or without viral load. The major strength
of the current study is that all the patients were during the acute/
clinical phase of chikungunya infection. The limitation of the pre-
sent study is the limited amount of DNA from the whole blood
samples of the patients during the outbreaks. This restrained us
from performing more detailed analyses of genes located close
to the HLA DQ-DR locus which would have helped in understand-
ing the underlying mechanism behind the newly found associa-
tions in the western Indian chikungunya patients.
In a study population of 101 patients with chikungunya infec-
tion and 104 healthy subjects from Rangat, Middle Andaman,
India, Chaaithanya et al. identified HLA-DQB1*03–03 allele and
some critical amino acid differences in the peptide binding pock-
ets of HLA-DQB1. They associated the same to have some role in
influencing infection and pathogenesis of chikungunya virus.26
At
the HLA class II locus, the frequency of DR7 has been reported to
be higher among Ross River virus patients than the controls.27
A study on chronic chikungunya patients has shown the preva-
lence of HLA-DRB1*01 and DRB1*04 alleles in patients who suf-
fered from rheumatoid arthritis (RA) after the infection.23
It has
been reported that HLA-DRB1SE (shared epitope) alleles are asso-
ciated with increased levels of anti-citrullinated protein antibodies
(ACPA) in different ethnic RA patients.28
The report that, unlike the
Malay and Chinese ACPA-positive RA patients, DRB1*04 is not a
significant allele among the ACPA-positive RA Indians goes in par-
allel with our observation.28
Lower allele frequencies of
HLA-DRB1*11 in the patient population of the current study sug-
gested its association with reduced risk towards CHIKV infection.
This goes in parallel with our previous report on association of
HLA-DRB1*11 allele in self-limiting hepatitis E patients from
Table 3. Statistical analysis of haplotypes association in patient vs control group
Haplotype DRB1*–DQB1* Patient (2n¼200)
n (%)
Control (2n¼500)
n (%)
Patient vs control
p-value OR (95% CI) Pc
15–06 50 (25.0) 125 (25.0) NS 1 (0.68–1.46) NS
10–05 16 (8.0) 39 (7.8) NS 1.028 (0.56–1.88) NS
07–02 14 (7.0) 38 (7.6) NS 0.91 (0.48–1.72) NS
14–05 22 (11.0) 33 (6.6) NS 1.7 (0.99–3.08) NS
11–03 2 (1.0) 30 (6) 0.007 0.15 (0.037–0.66) NS
04–03 20 (10.0) 27 (5.4) 0.042 1.94 (1.065–3.55) NS
07–03 11 (5.5) 19 (3.8) NS 1.473 (0.6881–3.155) NS
Human leukocyte antigen haplotype frequencies .5% in either group were only considered.
2n: total number of chromosomes; n: haplotype frequency; NS: not significant; Pc: Bonferroni corrected p-value.
Table 4. Statistical analysis of human leukocyte antigen (HLA)
allele associations in patient with viral load vs patient without viral
load group
HLA allele Patient
with viral
load
(2n¼34)
n (%)
Patient
without
viral load
(2n¼66)
n (%)
Patient with viral load vs
patient without viral load
p-value OR Pc
DQB1*02 6 (18) 7 (11) NS 1.8 (0.55–0 .87) NS
DQB1*03 9 (26) 19 (29) NS 0.8 (0.35–2.25) NS
DQB1*04 3 (9) ND NA NA NA
DQB1*05 7 (21) 15 (23) NS 0.7 (0.25–2.0) NS
DQB1*06 10 (30) 25 (38) NS 0.6 (0.28–1.66) NS
DRB1*01 2 (6) 3 (5) NS 1.3 (0.2–8.25) NS
DRB1*03 3 (9) 5 (8) NS 1.1 (0.26–5.26) NS
DRB1*04 2 (6) 6 (9) NS 0.6 (0.11–3.27) NS
DRB1*07 6 (18) 11 (17) NS 1.0 (0.35–3.19) NS
DRB1*10 1 (3) 5 (8) NS 0.3 (0.04–3.29) NS
DRB1*13 5 (12) 5 (8) NS 1.6 (0.4–6.5) NS
DRB1*14 3 (6) 6 (9) NS 0.6 (0.11–3.27) NS
DRB1*15 9 (32) 18 (27) NS 1.2 (0.51–3.13) NS
HLA allele frequencies .5% in either of the groups is considered.
2n: total number of chromosomes; n: allele frequency; NA: not
applicable; ND: not detected; NS: not significant; Pc: Bonferroni
corrected p- value.
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5. western Maharashtra.29
However, in a study from western India,
Amrapurkar et al. reported the association of DRB1*11 with viral
persistence in chronic hepatitis B infection.30
It may be possible
that the epitopes presented by HLA-DRB1*11 modulate the
immune response in western Indian patients with viral etiology.
Since this is the first report on association of HLA-DRB1 alleles
with CHIKV infection, in depth studies are needed to understand
the associated mechanism.
DQB1*04 allele only in chikungunya patients with viral load
suggests a possible association of the same with CHIKV
replication that may further be correlated with the viral clear-
ance/chronicity in a cohort of longitudinal study as reported by
de Almeida et al. in hepatitis C virus infection.31
In two locus haplotypes analysis, DRB1*11/DQB1*03 and
DRB1*04/DQB1*03 haplotypes revealed statistically significant
associations with resistance and susceptibility to CHIKV infection,
respectively. Although the former haplotype was six times more in
the control category than the patients, it was susceptible to
Bonferroni correction. Significantly higher prevalence of haplotype
HLA DRB1*11/DQB1*03 in hepatitis C virus antibody positive indi-
viduals from Maharashtra has been reported by our group.32
In an
HIV type 1 infected Botswana population, a two-locus haplotype
DRB1*04/DQB1*03, has been reported to be occurring at frequen-
cies above 10%.33
In a hospital based north Indian study of 31
cervical pre-cancer patients, DRB1*04/DQB1*03 haplotype exhib-
ited susceptibility to human papilloma virus mediated cervical
pre-cancerous lesions.34
No associations of DQB1*03, DRB1*04
alleles and an association of DRB1*04/DQB1*03 haplotype with
susceptibility to CHIKV infection in the current study population
might have emerged due to a strong linkage disequilibrium
between the individual alleles. At the haplotype end these are
important findings, since no report demonstrating the association
of DRB1/DQB1 haplotypes in chikungunya patients is available.
In conclusion, the emergence of HLA DRB1*11 as a resistant
allele provides meaningful clues on association of chikungunya
associated host genetic factors. Further, a longitudinal study
may define the putative role of HLA DRB1*11 in spontaneous
clearance of the virus/CHIKV associated chronicity/modulating
the CHIKV associated pathogenesis.
Authors’ contributions: AST and ST conceived the work. YG was
responsible for the clinical work and specimen collection. AH, ST and RD
conducted and interpreted the laboratory work. AST, ST, AH and RD
wrote the manuscript. All authors contributed to, and read and
approved the final manuscript. AST is guarantor for the paper.
Acknowledgments: We thank Director, National Institute of Virology for all
the encouragements. Special thanks are due to Mr Prasad Babbar, Mr Bipin
Tilekar, Mr Prakash B Jawalkar, Miss Neeta C Thorat, and Mr Shirish V
Vaidya, for technical help. Mr. Subrat Thanapati would like to thank the
University Grant Commission, New Delhi, India for providing the Junior
Research Fellowship.
Funding: The work was supported by the Indian Council of Medical
Research, New Delhi, India. The funding agency had no role in study
design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing interests: None declared.
Ethical approval: This study was approved by the Institutional Ethical
Committee for Research on Humans as per the guidelines of the Indian
Council of Medical Research.
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