Functional Single-Nucleotide Polymorphisms in the MBL2 and TLR3 Genes Influence Disease Severity in Influenza A (H1N1)pdm09 Virus-Infected Patients from Maharashtra,India

Abstract

The clinical outcome in influenza A (H1N1)pdm09 virus-infected subjects is determined by several factors, including host genetics. In the present study, single-nucleotide polymorphisms (SNPs) in the IFITM, MBL2, TLR3, TLR8, DDX58, IFIH1, CD55, and FCGR2, genes were investigated in influenza A (H1N1)pdm09 virus-infected subjects to find out their association with disease severity. Influenza A (H1N1)pdm09 virus-infected subjects with severe disease (n = 86) and mild disease (n = 293) from western India were included in the study. The SNPs were investigated by PCR-based methods. The results revealed a higher frequency of TLR3 rs5743313 T/T genotype [odds ratio (OR) with 95% confidence interval (CI) 2.55 (1.08–6.04) p = 0.039] and TLR3 two-locus haplotype rs3775291-rs3775290 T-A [OR with 95% CI 7.94 (2.05–30.68)] in severe cases. Lower frequency of the mutant allele of MBL2 rs1800450 [OR with 95% CI 0.51 (0.27–0.87), p = 0.01] and TLR3 two-locus haplotype rs3775291-rs3775290 T-G [OR with 95% CI 0.48 (0.27–0.85)] was observed in severe cases compared with cases with mild disease. Higher frequency of TLR3 two-locus haplotype rs3775291-rs3775290 T-A was observed in severe cases [OR with 95% CI 7.9 (2.0–30.7)]. The allele and genotype frequencies of other SNPs were not different between the study categories. The results suggest that the functional SNPs in MBL2 and TLR3 are associated with severe disease in influenza A (H1N1)pdm09 virus-infected subjects.

Introduction

Influenza A (H1N1)pdm09 virus causes severe acute respiratory illness and dominates the influenza season (30). During 2017, the Integrated Disease Surveillance Program, Government of India, has reported more than 2,000 deaths due to Influenza A (H1N1)pdm09 virus alone (18). Disease severity is influenced by several risk factors. Disease severity increases with age and highest risk of death rate has been observed in those above 50 years of age. Apart from age, obesity has also been identified as a risk factor for fatal outcome among those infected with Influenza A (H1N1)pdm09 virus (36). Host immune response and host genetics also impact the disease severity.

Many host proteins are known to directly interact with influenza viruses, whereas many other proteins participate in the immune response against influenza A (H1N1)pdm09 virus. Interferon-induced transmembrane protein 3 (IFITM3) is known to inhibit the replication of influenza A (H1N1)pdm09 virus (8). Mannose-binding lectin (MBL) is a protein, which interacts with multiple viruses (35). Apart from binding viruses, MBL activates complement pathway and promotes inflammatory response (13). The RNA genome of influenza A (H1N1)pdm09 virus is recognized by toll-like receptor (TLR)-3 and retinoic acid-inducible gene (RIG)-1 proteins.

Activation of TLR-3 and RIG-1 by the viral RNA culminates in type I interferon (IFN) response. Both TLR-3 and RIG-1 are required for maximum induction of type I IFN response by influenza A virus in lung cells (38). Myxoviral protein A (MxA) is known to inhibit influenza virus by interacting with virus nucleoprotein (27). CD55 is known to inhibit the complement and T cell-induced inflammation, which is an important factor in determining disease severity (9,42). Fcγ receptor IIA is a surface protein, which is known to perform multiple immune functions such as clearance of immune complex, phagocytosis, and regulating antibody function by binding to the Fc region of IgG antibody (3).

The availability and functioning of these host proteins are influenced by single-nucleotide polymorphisms (SNPs) in the genes that code for these proteins. SNPs in the genes that code for CD55, Fcγ receptor IIA, interleukin (IL)-1β, IL-10, and IL-17 have been shown to be associated with severe disease among Influenza A (H1N1)pdm09 virus-infected individuals (16,17,42,43).

A SNP in the IFITM3 gene (rs12252) has been shown to be associated with severe disease in influenza A (H1N1)pdm09 virus-infected subjects from different populations (39). Functional mutations in the coding region and SNPs in the promoter region have been investigated extensively for their association with susceptibility to various diseases (2,6,12). SNPs in the genes coding for TLR-3, MDA-5, RIG-1, and MxA have been shown to be associated with infectious disease due to ssRNA viruses (19,37,40). Our recent study has reported the association of a SNP in the TNFA gene with severe Influenza A (H1N1)pdm09 virus disease (5).

There is a lack of studies on the association between SNPs and susceptibility to severe influenza A (H1N1)pdm09 virus disease in India, where the virus has become seasonal and causes havoc due to consistently increased number of case fatalities. In the present study, the association between SNPs in the IFITM, MBL2, TLR3, TLR8, DDX58, IFIH1, CD55, and FCGR2A genes and severe influenza A (H1N1)pdm09 virus disease was investigated in influenza A (H1N1)pdm09-infected patients from Western India.

Materials and Methods

Study subjects

Patients with history of laboratory-confirmed influenza A (H1N1)pdm09 virus infection and/or ongoing treatment were contacted for enrollment in the study and blood samples were collected from the consenting study subjects. Patients with influenza A (H1N1)pdm09 virus infection admitted to the collaborating hospitals were also included in the study. A written informed consent was obtained from all the participants or their legal guardians before sample collection. The study was reviewed and approved by the Institutional Ethics Committee of ICMR-National Institute of Virology and the collaborating hospitals (NIV/IHEC/2013/D-1873 dated June 13, 2013).

Influenza A (H1N1)pdm09 virus infection in the study subjects during the acute phase of the disease was confirmed by real-time RT-PCR assay. DNA extraction from the blood samples was performed using a commercial DNA extraction kit (the QIAamp Blood DNA Midi Kit, Catalog no. 51185; Qiagen) according to the protocol given by the manufacturer. Case history sheets filled by the treating physicians were used to classify the patients into those with mild disease and severe disease. Patients admitted to intensive care unit and requiring ventilator support or the patients with fatal outcome were classified as those with severe Influenza A (H1N1)pdm09 virus disease. All the other patients were considered as those having mild disease (10,42).

Genotyping of SNP in TNFA, MBL2, and IFITM3 genes

TLR3 rs3775290 and rs5743313, MX1 rs2071430 and rs17000900, DDX58 rs10813831, MBL2 rs1800450 and rs1800451, and IFITM3 rs12252 polymorphisms were genotyped using PCR-RFLP-based methods as described earlier (4,15,32,40,41). Genotyping of MBL2 rs7096206 was performed using the PCR-SSP-based method as described earlier (34). TLR3 rs3775291, CD55 rs2564978, FCGR2A rs1801274, and IFIH1 rs1990760 were genotyped using KASP genotyping assays according to the manufacturer's instructions (LGC Biosearch Technologies, Petaluma, CA).

Statistical analysis

Statistical comparisons were carried out using online SNP stats program (33) or SPSS version 17. Allele frequency of each SNP was compared between the mild and severe cases using a 2 × 2 contingency table method test and the strength of the association was represented by odds ratio (OR) with 95% confidence intervals (CIs). Whether the genotype frequencies conform to Hardy–Weinberg equilibrium was investigated using chi-squared test. Haplotype frequencies were estimated using SNPstats. Logistic regression analysis, adjusted for age and gender, was performed to find out the association between genotypes/haplotypes and severe disease under different genetic models. A p-value of <0.05 was considered significant.

False discovery rate (FDR) was used to correct for multiple testing comparison using Benjamini–Hochberg method (FDR used 5%) (29). Power calculation was performed using G*Power software version 3.1.

Results

Demographic characteristics and disease profile of the study subjects

A total number of 379 influenza A (H1N1)pdm09 virus-infected patients from Pune and its adjoining districts, Maharashtra, Western India were included in the study. Among the patients, 86 were categorized as severe cases based on the requirement of ventilator support and/or fatal outcome. Forty patients had fatal outcome. Remaining 293 cases were categorized as having mild disease. The ratio of male to female is 1.15 for mild cases and 0.97 for severe cases. Severe cases had significantly higher age (mean ± standard deviation 50 ± 13) as compared with mild cases (mean ± standard deviation 39.1 ± 18) (p < 1*10^–6).

Allele frequencies of SNPs in IFITM, MBL2, TLR3, TLR8, DDX58, IFIH1, CD55, and FCGR2A, genes in influenza A (H1N1)pdm09 virus-infected patients with mild and severe disease

The frequency of the mutant allele of MBL2 rs1800450 was significantly lower in severe cases in comparison to cases with mild disease [OR with 95% CI 0.51 (0.27–0.87), p = 0.01]. However, the p-value was not significant after multiple testing comparisons (p > 0.05). The allele frequencies of other studied SNPs, including the IFITM3 rs12252 were not significantly different between the study categories (p > 0.05) (Table 1).

Table 1.

Allele Frequencies of IFITM3, CD209, MBL2, TLR3, DDX58, MX1, CD55, FCGR2A, and IFIH1 Gene Polymorphisms in Patients with Mild and Severe H1N1pdm09 Influenza Virus Infections

SNPs and alleles	Mild cases (2n = 586) N (%)	Severe cases (n = 172) N (%)	OR with 95% CIs	p
IFITM3 rs12252 T > C
T	504 (86.0)	154 (90.0)	—	—
C	82 (14.0)	18 (10.0)	0.7 (0.4–1.2)	0.28
MBL2 rs7096206 C > G
C	420 (72.0)	120 (70.0)	—	—
G	166 (28.0)	52 (30.0)	1.1 (0.74–1.61)	0.69
MBL2 rs1800450 C > T
C	482 (80.0)	155 (90.0)	—	—
T	106 (18.0)	17 (10.0)	0.5 (0.3–0.9)	0.01
MBL2 rs1800450 C > T
C	561 (96.0)	165 (96.0)	—	—
T	25 (4.0)	7 (4.0)	0.9 (0.3–2.3)	0.99
TLR3 rs3775290 G > A
G	399 (68.0)	117 (68.0)	—	—
A	187 (32.0)	55 (32.0)	1.0 (0.7–1.5)	0.99
TLR3 rs3775291 C > T
C	436 (75.0)	134 (78.0)	—	—
T	148 (25.0)	38 (22.0)	0.8 (0.5–1.2)	0.44
TLR3 rs5743313 C > T
C	434 (74.0)	120 (70.0)	—	—
T	152 (26.0)	52 (30.0)	1.2 (0.8–1.8)	0.31
DDX58 rs10813831 G > A
G	495 (84.0)	141 (82.0)	—	—
A	91 (16.0)	31 (18.0)	1.2 (0.7–1.9)	0.50
MX1 rs17000900 C > A
C	460 (78.0)	132 (77.0)	—	—
A	126 (22.0)	40 (23.0)	1.1 (0.7–1.7)	0.69
MX1 rs2071430 G > T
G	378 (65.0)	100 (58.0)	—	—
T	208 (35.0)	72 (42.0)	1.3 (0.9–1.9)	0.15
CD55 rs2564978 C > T
C	322 (59.0)	106 (62.0)	—	—
T	220 (41.0)	64 (38.0)	0.8 (0.6–1.2)	0.45
FCGR2A rs1801274 A>G
A	338 (58.0)	99 (58.0)	—	—
G	248 (42.0)	71 (42.0)	1.0 (0.7–1.4)	0.90
IFIH1 rs1990760 C > T
C	317 (55.0)	83 (48.0)	—	—
T	263 (45.0)	89 (52.0)	1.1 (0.8–1.5)	0.69

Bold values represent significant values.

CI, confidence interval; OR, odds ratio; SNP, single-nucleotide polymorphism.

Genotype frequencies of SNPs in IFITM, MBL2, TLR3, TLR8, DDX58, IFIH1, CD55, and FCGR2A genes in influenza A (H1N1)pdm09 virus-infected patients with mild and severe disease

The genotype frequencies did not deviate from Hardy–Weinberg equilibrium in both mild and severe cases (p > 0.05), except for MX1 rs2071430 in mild cases (p = 0.03). The carrier frequency of MBL2 rs1800450 mutant allele containing genotypes was lower in severe cases compared with subjects with mild disease. However, the OR [OR with 95% CI 0.54 (0.29–1.00), p = 0.042] was not significant. The mutant allele of MBL2 rs1800450 was significantly associated with disease severity in log-additive model [OR with 95% CI 0.55 (0.32–0.96) p = 0.024].

The T/T genotype of TLR3 rs5743313 was associated with severe disease in recessive mode (T/T vs. C/C+C/T) [OR with 95% CI 2.55 (1.08–6.04), p = 0.039]. However, the p-values were not significant after multiple testing comparisons (p > 0.05). The genotype frequency distributions of other studied SNPs between mild and severe cases were comparable (p > 0.05) (Table 2).

Table 2.

Genotype Frequencies of IFITM3, CD209, MBL2, TLR3, DDX58, MX1, CD55, FCGR2A, and IFIH1 Gene Polymorphisms in Patients with Mild and Severe H1N1pdm09 Influenza Virus Infections

SNPs and genotypes	Mild cases (n = 293) N (%)	Severe cases (n = 86) N (%)	OR with 95% CIs	p
IFITM3 rs12252 T > C
T/T	214 (73.0)	68 (79.1)	Ref.
T/C	76 (25.9)	18 (20.9)	0.7 (0.4–1.2)	0.15
C/C	3 (1.0)	0	0
MBL2 rs7096206 C > G
C/C	152 (51.9)	42 (48.8)	Ref.
C/G	116 (39.6)	36 (41.9)	1.2 (0.7–2.1)	0.75
G/G	25 (8.5)	8 (9.3)	1.1 (0.4–2.7)
MBL2 rs1800450 C > T
C/C	199 (67.9)	70 (81.4)	Ref.
C/T	82 (28.0)	15 (17.4)	0.6 (0.3–1.1)	0.071
T/T	12 (4.1)	1 (1.2)	0.2 (0–1.7)
C/T+T/T	94 (32.1)	16 (18.6)	0.5 (0.3–1.0)	0.043
Log-additive model	—	—	0.5 (0.3–0.9)	0.024
MBL2 rs1800450 C > T
C/C	268 (91.5)	79 (91.9)	Ref.
C/T	25 (8.5)	7 (8.1)	1.3 (0.5–3.2)	0.59
T/T	0	0	NA
TLR3 rs5743313 C > T
C/C	160 (54.6)	44 (51.2)	Ref.
C/T	114 (38.9)	32 (37.2)	0.9 (0.5–1.6)	0.12
T/T	19 (6.5)	10 (11.6)	2.5 (1.1–6.0)	0.039
TLR3 rs3775291 C > T
C/C	169 (57.9)	55 (64.0)	Ref.
C/T	98 (33.6)	24 (27.9)	0.8 (0.4–1.4)	0.62
T/T	25 (8.6)	7 (8.1)	0.8 (0.3–1.9)
TLR3 rs3775290 G > A
G/G	138 (47.1)	36 (41.9)	Ref.
G/A	123 (42.0)	45 (52.3)	1.4 (0.8–2.4)	0.19
A/A	32 (10.9)	5 (5.8)	0.6 (0.2–1.8)
DDX58 rs10813831 G > A
G/G	208 (71.0)	57 (66.3)	Ref.
G/A	79 (27.0)	27 (31.4)	1.3 (0.7–2.2)	0.71
A/A	6 (2.0)	2 (2.3)	1.2 (0.2–6.6)
MX1 rs17000900 C > A
C/C	176 (60.1)	48 (55.8)	Ref.
C/A	108 (36.9)	36 (41.9)	1.2 (0.7–2.0)	0.59
A/A	9 (3.1)	2 (2.3)	0.5 (0.1–3.0)
MX1 rs2071430 G > T
G/G	113 (38.6)	27 (31.4)	Ref.
G/T	152 (51.9)	46 (53.5)	1.2 (0.7–2.0)	0.44
T/T	28 (9.6)	13 (15.1)	1.7 (0.8–3.9)
CD55 rs2564978 C > T
C/C	107 (36.5)	34 (40.0)	Ref.
C/T	131 (44.7)	38 (44.7)	0.9 (0.5–1.5)	0.43
T/T	55 (18.8)	13 (15.7)	0.6 (0.3–1.3)
FCGR2A rs1801274 A > G
A/A	104 (35.5)	28 (32.9)	Ref.
A/G	130 (44.4)	43 (50.6)	1.3 (0.7–2.3)	0.47
G/G	59 (20.1)	14 (16.5)	0.9 (0.4–1.9)
IFIH1 rs1990760 C > T
C/C	87 (30.0)	19 (22.1)	Ref.
C/T	143 (49.3)	45 (52.3)	1.5 (0.8–2.7)	0.43
T/T	60 (20.7)	22 (25.6)	1.4 (0.7–3.0)
TLR8 rs3764879 C > G Males
C	75 (52.4)	24 (53.3)	—
G	68 (47.6)	21 (46.7)	0.8 (0.4–1.6)	0.50
Females
G/G	42 (28.6)	15 (38.5)	Ref.
G/C	75 (51.0)	15 (38.5)	0.5 (0.2–1.2)	0.28
C/C	30 (20.4)	9 (23.1)	0.7 (0.2–1.9)

Bold values represent significant values.

Haplotype frequencies of MBL2 and TLR3 gene SNPs and in influenza A (H1N1)pdm09 virus-infected patients

MBL2 rs7096206- rs1800450- rs1800451 haplotype C-A-G was significantly lower in severe cases as compared with mild cases [OR with 95% CI 0.50 (0.26–0.95) p = 0.035]. However, the p-value was not significant after multiple testing comparisons (p > 0.05). Other MBL2 haplotypes were not significantly different between severe and mild cases.

Among the TLR3 three- and two-locus haplotypes (rs5743313-rs3775291-rs3775290 and rs3775291-rs3775290), the frequency of haplotypes C-T-A [OR with 95% CI 9.73 (2.38–39.79)] and T-A [OR with 95% CI 7.94 (2.05–30.68)] were higher in severe cases suggesting their association with severe disease. The p-values remained significant even after multiple test comparisons (p = 0.0174). The frequency of TLR3 two-locus haplotype T-G was lower in severe cases [OR with 95% CI 0.48 (0.27–0.85)] suggesting its association with protection to severe disease (Table 3). However, the p-values were not significant after multiple testing comparisons (p > 0.05).

Table 3.

Haplotype Frequencies of MBL2 and TLR3 Gene Polymorphisms in Patients with Mild and Severe H1N1pdm09 Influenza Virus Infections

SNPs and haplotypes	Mild cases (%)	Severe cases (%)	OR with 95% CIs	p
MBL2 haplotypes rs
C-C-C	50.1	58.9	Ref.	—
G-C-C	27.5	27.9	0.9 (0.6–1.4)	0.62
C-T-C	17.6	9.1	0.5 (0.3–0.9)	0.035
C-C-T	4.0	1.0	0.6 (0.1–3.0)	0.53
TLR3 three locus haplotypes rs5743313-rs3775291-rs3775290
C-C-A	28.7	25.7	Ref.	—
T-C-G	22.2	29.2	1.6 (0.9–2.7)	0.078
C-T-G	22.8	16.9	0.7 (0.4–1.3)	0.25
C-C-G	21.5	21.9	1.1 (0.6–2.0)	0.77
T-C-A	2.2	1.0	0.5 (1.0–4.9)	0.57
C-T-A	0.9	5.2	9.7 (2.4–39.8)	0.0017
T-T-G	1.5	0	0	1
TLR3 two-locus haplotypes rs3775291-rs3775290
C-G	43.8	51.5	Ref.	—
C-A	30.9	26.4	0.7 (0.4–1.1)	0.14
T-G	24.3	16.5	0.5 (0.3–0.8)	0.013
T-A	1.0	5.5	7.9 (2.0–30.7)	0.0029

Bold values represent significant values.

Discussion

In the present study, the results indicated the association of MBL2 rs1800450 mutant allele (T), T allele containing MBL2 rs1800450 genotypes, and TLR3 rs3775291-rs3775290 T-G haplotype with protection against severe disease. Furthermore, the results revealed that the TLR3 rs5743313 T/T genotype and TLR3 rs3775291-rs3775290 T-A haplotype were associated with severe disease.

MBL2 rs1800450 leads to the substitution of glycine with aspartic acid in the 54th position of MBL protein. As a result, oligomerization of MBL monomer is affected leading to deficiency of functional MBL levels in plasma (11). Association of both higher levels as well as deficiency of MBL with susceptibility to infectious diseases, including disease severity in influenza virus-infected subjects has been reported (1,14,20).

Influenza A (H1N1)pdm09 virus is resistant to neutralization by MBL due to the presence of only one glycosylation site in the head of the hemagglutinin as compared with the presence of three to four glycosylation sites in other influenza A viruses (16). MBL has been shown to bind and promote clearance of seasonal influenza A viruses and dampen host inflammatory response in alveolar macrophages (26). Although MBL can bind influenza A (H1N1)pdm09 virus, due to less number of glycosylation sites in the virus, the binding may not be sufficient to prevent infection of the target cells and generation of subsequent inflammatory response (21).

In the absence of sufficient levels of functional MBL, inflammatory response might be blunted. Thus, it is plausible that MBL2 alleles associated with deficiency of functional MBL might be associated with protection to severe disease. However, in the present study, no association was observed with MBL2 rs1800451, which also leads to deficiency of functional MBL. The frequency of MBL2 rs1800451 mutant allele is too low to detect an association with disease severity due to the smaller sample size.

Similarly, no association was also observed between MBL2 rs7096206 and influenza A (H1N1)pdm09 virus disease severity. MBL2 rs7096206 affects transcription and leads to low levels of MBL but does not affect oligomerization of MBL (11). Low levels of functional MBL might still induce inflammatory response and hence it is possible that MBL2 rs7096206 may not be associated with disease severity.

The current study also revealed the association of TLR3 SNPs with severe influenza A (H1N1)pdm09 virus disease. TLR3 rs5743313 T/T genotype was associated with severe disease in the recessive mode. This SNP is located in the intronic region near exon 4 and might contribute to altered TLR-3 signaling, thereby affecting disease susceptibility.

Contrasting to the present study, a study on Chinese subjects with H1N1 or H7N9 infections reported the association of TLR3 rs5743313 C/C genotype with fatal cases [19]. Another study on European children reported the association of TLR3 rs5743313 C/T genotype with increased risk of pneumonia in H1N1-infected children (7). The discrepancies between the studies might be due to the ethnicity-specific differences. TLR3 rs3775291-rs3775290 two-locus haplotypes T-A and T-G showed contrasting associations suggesting the role of combination of rs3775291 and rs3775290 SNPs in influencing susceptibility to severe disease.

Both rs3775291 and rs3775290 are present in the coding region and rs3775291 substitute phenyl alanine for leucine, whereas rs3775290 substitutes leucine for phenyl alanine at the 412th and 459th positions in the TLR-3 protein, respectively. TLR3 rs3775291 T allele has been shown to be associated with reduced proinflammatory cytokine response in response to cytomegalovirus stimulation of peripheral blood mononuclear cells (25). It is possible that in the background of rs3775291 T allele, rs3775290 alleles might contribute to increased or decreased TLR-3 signaling and hence contrasting associations of rs3775291-rs3775290 T-A and T-G haplotypes with severe influenza A (H1N1)pdm09 virus disease.

Multiple studies have shown the association of IFITM3 rs12252 with influenza disease severity. The mutant allele (C) of IFITM3 rs12252 has been shown to be associated with severe disease in a European population (8). However, this was not confirmed in another study carried out in Europeans, which suggested the association of IFITM3 rs12252 with mild influenza (24). Studies from Chinese patients reported the association of rs12252 C allele with disease severity in influenza A-infected subjects, including the influenza A (H1N1)pdm09 virus (19,28,41). A Study from European population reported no association of rs12252 with influenza A (H1N1)pdm09 virus disease severity (22).

In the present study, no association between IFITM3 rs12252 and influenza A (H1N1)pdm09 virus disease severity was observed. The minor allele frequency reported in this population is higher than in European population but less than in Chinese population (34,41). IFITM3 rs12252 is a variant affecting splicing of transcripts leading to a truncated protein lacking first 21 amino acids (8).

However, recent studies have reported that subjects with IFITM3 rs12252 C allele still produce full-length protein (23). Although the genotype frequency distributions are in Hardy–Weinberg equilibrium, still subjects homozygous for the C allele were observed only in the mild cases group in the present study and the sample size might be low to detect an association. However, the sample size is comparatively higher than the previous studies. Moreover, TLR8, MX1, DDX58, IFIH1, CD55, and FCGR2A genes showed a lack of association with disease severity in influenza A (H1N1)pdm09 virus-infected subjects suggesting their limited or redundant functions in the immune response against the virus.

Except for TLR3 haplotypes, association of MBL2 alleles, genotypes, and haplotypes with severe disease lost its significance after multiple testing comparisons. This could be due to the lower sample size of severe cases. The sample size had a power of more than 80% to detect the observed difference in the frequency of MBL2 rs1800450 C/T+ TT genotypes between mild and severe cases. To conclude, the present study suggest that SNPs in the MBL2 and TLR3 were associated with disease severity in influenza A (H1N1)pdm09 virus-infected patients. These SNPs can be targeted for predicting severe disease and further understanding of the role of MBL and TLR-3 proteins in influenza A (H1N1)pdm09 virus disease, and how the SNPs regulate functioning and levels of these proteins might have implications for designing therapeutic strategies against severe disease.

Availability of Data and Material

All data have been provided in the article.

Ethics Approval

The study was approved by the Institutional Human Ethics Committees of the ICMR-NIV and collaborating hospitals (NIV/IHEC/2013/D-1873 dated June 13, 2013).

Consent to Participate

A written informed consent was provided by the study subjects or their legal guardians.

Consent for Publication

The article was approved by all authors and the Institutional Article Review Committee.

Footnotes

Authors' Contributions

K.A. and M.L.C. conceived and designed the project. M.L.C., U.C., M.S., and P.S. did the work. V.S.P., S.A.S., S.S., A.R.B., and P.D. were involved in recruitment of patients. K.A. did the statistical analysis. K.A. and M.L.C. wrote the article. All authors approved the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

The study was funded by a grant from the Department of Biotechnology, Government of India (BT/PR8957/MED/12/630/2013 dated 08.02.2017) to Dr M.L.C.

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