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13328

Immunogenetic Study of Human RHINOVIRUS and TLR 1 AND TLR 2 Gene Polymorphism in Children and Infants Suffering from Acute Respiratory Tract

Infection

ALI ABDUL AMEER SHAHEED1*AND SHAKIR H. MOHAMMED AL-ALWANY2

1College of Science, Babylon University, Iraq. 2*College of Science, Babylon University, Iraq.

1*[email protected] Abstract.

Background: Acute respiratory infections (ARI) are the leading cause of mortality in children under five years of age worldwide and most of these deaths are due to bronchiolitis and pneumonia.

Recent evidence from studies using genome detection systems such as polymerase chain reaction or micro-array technology show that, in most cases, these deaths are caused or precipitated by viruses.

Many studies showed that RV is one of the leading causes of pneumonia, bronchiolitis and other form of severe respiratory disease, standing side by side with respiratory syncytial virus (RSV) in children and influenza in elderly. TLR 1 (rs5743557) and TLR2 (rs1898830) gene polymorphism may be as risk factor for RTI. Objective: Detection of the HRV and TLR1 and TLR2 gene polymorphism in Iraqi patients less than 10 years which suffering from RTI.

Patients and Methods: 100 specimens including: 75 specimens of children suffering from RTI &

25 specimens from apparently healthy children as a control group. Nasopharyngeal and/or throat swabs as well as blood (whole blood & serum) samples were collected and processed for screened for human RHINOVIRUS (HRV) and TLR1 and TLR2 gene polymorphism.

Results: HRV was detected 56% (28out of 50 cases) by using RT PCR. No HRV-signals were reported in the control samples. The results of genetic Polymorphism of TLR 1 (rs5743557) and TLR2 (rs1898830) were 64% (48out of 75 patients) and 50.7% (38 out of 75) positive signal respectively by PCR. Statistically, A strong positive relationship (with highly Sig. correlation) was found between HRV and TLR 1 (rs5743557) and TLR2 (rs1898830) gene polymorphism in ages patients have ARTI.

Conclusions: human rhinovirus might be role in the respiratory tract infection and TLR1 and TLR2 gene polymorphism as risk factor in the Iraqi infants and young children patients suffering from severe RTI.

Keywords. RTI, HRV, RT PCR, TLR 1 (rs5743557), TLR2 (rs1898830).

INTRODUCTION

Acute respiratory tract infection (ARTI) is a leading cause of fever and hospitalization in children worldwide. Upper respiratory tract infection (URTI) is the most frequent human illness and the most common disease in childhood; on average, infants and preschoolers develop six to eight URTIs per year. URTIs can cause non-severe but wide-spread epidemics that are responsible for the continuous circulation of pathogens in the community (1).

For both URTI and LRTI, viral infection is the primary cause, especially in children. Multiple

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viral infections were reported to be associated with increased hospital admissions, intensive care unit admissions, lengthened duration of hospitalization, and prolonged mechanical ventilation use(2).

Rhinoviruses (RVs) are small, non-enveloped viruses that belong to the family Picornaviridae, genus Enterovirus. To date, there are 171 rhinovirus (RV) genotypes recognized and classified into the three species as RV-A (83 types), RV-B (32 types), and RV-C (56 types). RV-A and RV-B were discovered by isolation on monkey kidney cells in 1950s, while RV-C genotypes, which are not cultivable using ordinary culture methods, have been identified decades after following the rise of molecular techniques(3).

Toll-like receptors (TLRs) are important Pattern-recognition receptors (PRRs) are a group of innate conserved sensors, which actively contribute to detection of exogenous or endogenous molecules derived from microbes or from host cells. Among PRRs, TLRs play a role in allergic diseases since their relevance in asthma is well documented. TLRs ligands derived from pathogens or from host are known as pathogen-associated molecular patterns or damage associated molecular patterns, respectively(4).

In the respiratory epithelial cells, TLRs 1, 2, 4, 6 and 10 are expressed on the cell surfaces. TLR2 is expressed on the surface of immune cells and tissues as a heterodimer complex with either TLR1 or TLR6.TLR2 has a critical role in recognizing many viral(5).

MATERIALS ANDMETHODS

This prospective-control study was done for a seventy –five patients and twenty-five apparently healthy children as a control group including infants and children up to six months to ten years of age, whom admitted for RTI in Central Teaching Hospitals of Pediatric of Baghdad, Child Protection Teaching Hospital - Medical City and Babylon hospital teaching for pediatric and Gynecology as well as private laboratories. The specimens were collected during winter from November, 2020 to March, 2021. Nasopharyngeal and/or throat swabs as well as blood (whole blood & serum) samples were collected and processed for screened for human RHINOVIRUS (HRV) and TLR1 and TLR 2 genepolymorphism.

Nasopharyngeal and/or throat swabs as well as blood (whole blood & serum) samples were collected and processed for screened for human RHINOVIRUS (HRV) and TLR1 and TLR 2 genepolymorphism.

A special kit (PowerChek™ Rhinovirus Real-time PCR Kit) was used by the RT PCR device to detect the presence of the HRV virus in the 50 samples that gave a positive result in gel electrophoresis.

Detection of SNPs TLR 1 (rs5743557) and TLR2 (rs1898830) in whole blood samples of patients and AHC in several stages. DNA product to detect SNP (TLR 1 (rs5743557) and TLR2 (rs1898830)) by ARMS technique.

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13330 Primers sets that used for detection of TLR-1&2 polymorphism.

These primers designed especially for this study where the PCR device was used to amplify the intended SNPs and then use the gel electrophoresis technique to indicate the movement of the SNPs inside the gel.

SPSS program (Version– 23) &P value was considered Sig. when p <0.05 was used to explain the statistical variable between study populations.

RESULTS

Age Frequency of Patients with RTI and Healthy Control Groups

The age mean of the patients with (RTI) were (40.56 months) was less than the mean age of the apparently healthy control (AHC) (43.6months). There are non-Sig. statistical differences (p=0.47) between patients and apparently healthy control groups (AHC), show in table 1.

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Table1.FrequencyofPatientswithRTIandHealthyControlGroupsAccordingtotheir Age

Distribution of Patients with Respiratory Tract Infection According to Age Stratum

Figure (1) shows the age stratum of the study population groups. The age group 6-44 months was the predominant group with 47% and 16% followed by 45-83 month (16%) and (7%) finally 84-122 month (9%) and (6%) for patients and AHC respectively.

.Figure 1. Age Groups of the Study Population

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13328 Detection of HRV by Molecular Technique (Extraction Nucleic Acid by Specific Viral DNA/RNA Extraction Kit (Intron/Korea)

By using specific viral DNA/RNA extraction kit (Intron/Korea); the viral genomic was extracted , purifying and migrated using agarose gel from the swabs and blood of infants and adult children's patients with respiratory tract infection as a first step to amplify the target HRV-RNA.

Out of 100 NPS\TS and blood specimens involved in this study 50 (50%) were found to have a viral infection; including (50) patients equivalent to 66.7% of the total (75) patients with ARTI who were sampled in this study, more than patients who did not show a Viral genome.

While No viral nucleic acid was detected among all the examined healthy specimens (25) as control group as shown in Figures (3). There were statistically Sig. differences (p = 0.03) between patients with the viral genome and those without the Viral genome Table (3).

Table3.PercentageofViralGenomeExtractionofAcuteRespiratoryInfectionPatients and Apparently Healthy ControlGroups

Viral Geno me

Study Groups AHC

No.(

25)

ARTI No.(7 5)

Chi-Square (P-value) P

o si ti v e

N 0 50

P=0.03 Sign.

(P<0.05)

% 0% 66.7

%

N e g a ti v e

N 25 25

% 100

%

33.3

%

T o t a l

N 25 75

% 100

%

100%

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Figure 3. Extraction Viral Genome. 1 % Agarose Gel Electrophoresis, TBE 1X , 75 v for 60 min. M: Refers to DNA Size Marker 100-1100

DETECTION AND QUANTIFICATION OF HRV GENOME WITH REAL TIME PCR The quantity HRV system is a quantitative test that allows the DNA amplification and quantification, by means of Real-Time PCR, of HRV genome. The analysis of results was made by using a Real-Time PCR analyzer (Rotor-gene Q MDX/thermal cycler integrated with a system for fluorescence detection and a dedicated software). The positive result according to RT-PCR shows 56% (28 of 50 cases) as positive while 44% (22 of 50 cases) as negative, these results show in the Table (4) and Figure(4.A).

Table4.PercentageofPatientswithRTIandHealthyControlGroupsAccordingtotheir Age

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13330 Figure 1.A: Detection of HRV by RT-PCR

DETECTION OF SNPS TLR 1 (rs5743557) and TLR2 (rs1898830) GENE POLYMORPHISMS BY PCR.

Were positive results of SNPs TLR 1 (rs5743557) and TLR2 (rs1898830) gene polymorphisms by PCR detection in infants and adult children's patients with RTI as well AHC groups, where 64% (48 out of 75 cases) showed positive signals of TLR 1 (rs5743557), while, positive SNPs ofTLR2 (rs1898830) –PCR (constituting 50.7% 38 out of 75 cases) Figure (5 A,B) Table (5 A,B).

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Figure 5.ASNPs OF TLR 1 (rs5743557) Gene Polymorphism by PCR

Figure 5.B SNPs OF TLR2 (rs1898830) Gene Polymorphism by PCR

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13332 (TABLE. 5 .A) Genotype Distribution and Odd Ratio of TLR.1 Gene Polymorphisms

Between the ARTI Patients andAHC.

The result of amplified was appeared the presence of two bands (G Allele= 176 bp and A Allele=

233bp (due to the presence of the G>A mutation. Whereas the wild type was identified by a single 409 bp fragment.

Itcan be seen that the frequency of GG and AG

genotypes in patients with ARTI and AHC

groups which reached 27.1%, 20.8% ;16.7% and 25%,

respectively. It wassignificantly

increased in patients than control, in which GG

genotype increased as rate OR=2.452 compared with

GA and AA genotypes in studied groups. On the other

hand, the frequency of AA genotype in patients with ARTI and AHC

groups were 52.1%

and 58.3% respectively, that decreased in patients

compared with control group.

From the table (5.A) showed the statically analysis found a high significant inTLR.1 gene polymorphism of that Iraqi patient.

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(TABLE. 5 B) Genotype Distribution and Odd Ratio of TLR.2 Gene Polymorphisms Between the ARTI Patients andAHC.

The result of amplified was appeared the presence of two bands (G Allele= 163 bp and A Allele=

109bp ( due to the presence of the G>A mutation. Whereas the wild type was identified by a single 272 bp fragment.

It can be seen that the frequency of GG and AG

genotypes in patients with ARTI and AHC

groups which reached 31.8%, 23.7%, 20%; and 20% respectively. It was significantly increased in

patients than control, in which GG

genotype increased as rate OR=2.679 compared with

GA and AA genotypes in studied groups. On the other

hand, the frequency of AA genotype in patients with ARTI and AHC

groups were 44.7%

and 60% respectively, that decreased in patients

compared with control group.

From the table (5.B) showed the statically analysis found a high significant in TLR.2 gene polymorphism of that Iraqi patient.

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13334 DISCUSSION

Rhinovirus (RV) is the most common cause of viral upper respiratory illness resulting in rhinitis, sinusitis, pharyngitis, or otitis media, and can lead to the development of bacterial superinfections.

While most individuals only experience mild symptoms during a RV infection, children, the elderly, the immunosuppressed, and those with asthma, COPD, or cystic fibrosis are predisposed to lower respiratory tract illnesses including wheezing, asthma exacerbations, and respiratory distress that can often result in hospitalization. RV has also been implicated in the initiation of asthma, as the presence of wheeze during acute RV infection in the first few years of life is strongly correlated with the development of asthma later in life.(6)

The positive result according to RT-PCR shows 56% (28 of 50 cases) as positive while 22% (22 of 50 cases) as negative, these result shows in the Table (4). These results are converging with samples taken from children for HRV detection in Saudi Arabia, Jordan, and Egypt, (20%), (72.3%), and (35.2%), respectively. (7) (8)

HRVs were mainly associated with lower RTIs (in the absence of other viral agents) and wheezing in immunocompetent pediatric patients‎. the children who were positive for RV viremia simultaneously had a significantly higher viral load and more severe disease, thus suggesting that the former is a prerequisite for viremia and is more frequently associated with greater clinical involvement. (9)

RV transmission generally occurs through direct exposure to, and inhalation of, respiratory droplets/micro-droplets, even though it can also take place via fomites (contaminated surfaces), including direct person-to-person contact, since RV has moderate resistance to alcohol hand rubs and to other common disinfectants.(10)

In our current study we found the amplified of TLR-1 (rs5743557) target sequences appear by the presence of two bands G Allele= 176 bp and A Allele= 233bpdue to the presence of the G>A mutation.

The frequency of GG in patients and in (AHC) 23 out of 75 case (30.7%), 5 out of 25 case (20%) respectively that showed clearly increasing in patients than AHC and its odd ratio 2.489 which means the effect is associated with increased incidence of the outcome.

The frequency of AG in patients and in (AHC) 18 out of 75 case (24%), 2 out of 25 case (8%) respectively that showed clearly increasing in patients than AHC and its odd ratio 0.234 which means the effect is associated with lower incidence of the outcome.

The frequency of AA in patients and in (AHC) 34 out of 75 case (45.3%), 18 out of 25 case (72%) respectively that showed decreasing in patients than AHC and its odd ratio 0.551 which means the effect is associated with lower incidence of the outcome.

polymorphisms in TLRs can influence the immune response (susceptibility or resistance) of an

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individual to a pathogenic infection principally by affecting the host‐ pathogen interactions.

Intriguingly. (11)

In our current study we found the amplified of TLR-2 (rs1898830) target sequences appear by the presence of two bands G Allele= 163 bp and A Allele= 109bp ( due to the presence of the G>A mutation.

The frequency of GG in patients and in (AHC), 17 out of 75 case (22.7%), 6 out of 25 case (24%) respectively that showed clearly increasing in patients than AHC and its odd ratio 2.452 which means the effect is associated with increased incidence of the outcome.

The frequency of AG in patients and in (AHC), 19 out of 75 case (25.3%), 3 out of 25 case (12%) respectively that showed clearly increasing in patients than AHC and its odd ratio 0.321 which means the effect is associated with lower incidence of the outcome.

The frequency of AA in patients and in (AHC), 39 out of 75 case (52%), 16 out of 25 case (64%) respectively that showed decreasing in patients than AHC and its odd ratio 0.551 which means the effect is associated with lower incidence of the outcome.

TLR2 is a key regulator of host immunity and plays a pivotal role in deciding the fate of several microbial andR677W is considered as the most common TLR2‐ SNP that affects pathogen recognition ability of TLR2 and dampens antipathogenic host responses. (12)

REFERENCES

1- Yen, C. Y., Wu, W. T., Chang, C. Y., Wong, Y. C., Lai, C. C., Chan, Y. J., ... & Hung, M. C.

(2019). Viral etiologies of acute respiratory tract infections among hospitalized children–A comparison between single and multiple viral infections. Journal of Microbiology, Immunology and Infection, 52(6), 902-910.

2- Galanti, M., Birger, R., Ud‐Dean, M., Filip, I., Morita, H., Comito, D., ... & Shaman, J. (2019).

Longitudinal active sampling for respiratory viral infections across age groups. Influenza and other respiratory viruses, 13(3), 226-232.

3- Pan, C. Y., Yagi, S., Padilla, T., Ng, T. F. F., Marine, R. L., Nix, W. A., & Wadford, D. A.

(2018). Genome sequences of rhinovirus genotype C56 detected in three patients with acute respiratory illness, California, 2016 to 2017. Microbiology resource announcements, 7(7).

4- Zakeri, A., & Russo, M. (2018). Dual role of toll-like receptors in human and experimental asthma models. Frontiers in immunology, 9, 1027.

5- Henrick, B. M., Yao, X. D., Taha, A. Y., German, J. B., & Rosenthal, K. L. (2016). Insights into

(13)

13336 soluble Toll-like receptor 2 as a downregulatory of virally induced inflammation. Frontiers in immunology, 7, 291.

6- Griggs, T. F., Bochkov, Y. A., Basnet, S., Pasic, T. R., Brockman-Schneider, R. A., Palmenberg, A. C., & Gern, J. E. (2017). Rhinovirus C targets ciliated airway epithelial cells. Respiratory research, 18(1), 1-11.

7- Lee, W. M., Lemanske Jr, R. F., Evans, M. D., Vang, F., Pappas, T., Gangnon, R., ... & Gern, J.

E. (2012). Human rhinovirus species and season of infection determine illness severity. American journal of respiratory and critical care medicine, 186(9), 886-891.

8- Gad, M. N., Refaay, D., Gad, N., & Mohamed, A. Z. (2017). Viral infections in Egyptian hospitalized children with acute respiratory tract infections. J Clin Cell Immunol, 8, 526.

9- Esposito, S., Daleno, C., Scala, A., Castellazzi, L., Terranova, L., Papa, S. S., ... & Principi, N.

(2014). Impact of rhinovirus nasopharyngeal viral load and viremia on severity of respiratory infections in children. European journal of clinical microbiology & infectious diseases, 33(1), 41-48.

10- Esposito, S., Daleno, C., Tagliabue, C., Scala, A., Tenconi, R., Borzani, I., ... & Principi, N.

(2012). Impact of rhinoviruses on pediatric community-acquired pneumonia. European journal of clinical microbiology & infectious diseases, 31(7), 1637-1645.

11- Mukherjee, S., Huda, S., & Sinha Babu, S. P. (2019). Toll‐like receptor polymorphism in host immune response to infectious diseases: A review. Scandinavian journal of immunology, 90(1), e12771.

12- Mukherjee, S., Huda, S., & Sinha Babu, S. P. (2019). Toll‐like receptor polymorphism in host immune response to infectious diseases: A review. Scandinavian journal of immunology, 90(1), e12771.

13- Ullah Khan , F. ., & Hussain , N. . (2020). NH Serological and Molecular Based Diagnosis of Toxoplasma gondii in Galliformes by using ToxPK1 gene. Journal of Scientific Research in Medical and Biological Sciences, 1(2), 116-122. https://doi.org/10.47631/jsrmbs.v1i2.58

14- Abdel-Moneim AS, Kamel MM, Al-Ghamdi AS, Al-Malky MI (2013). Detection of bocavirus in children suffering from acute respiratory tract infections in Saudi Arabia. PloS one 8(1):e55500.

15- Chuang CY, Kao CL, Huang LM, Lu, CY, Shao PL, Lee PI, Chang LY (2011). Human bocavirus as an important cause of respiratory tract infection in Taiwanese children. Journal of Microbiology, Immunology and Infection 44(5):323-327.

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16- MunitzA,BrandtEB,MinglerM,FinkelmanFD,RothenbergME(2008).DistinctrolesforIL- 13and IL-4 via IL- α -4 receptor in asthma pathogenesis. Proceedings of the National Academy of Sciences 105(20):7240-7245.

17- Melendi GA, Laham FR, Monsalvo AC, Casellas JM, Israele V, Polack NR, Polack FP (2007).

Cytokine profiles in the respiratory tract during primary infection with human metapneumovirus, respiratory syncytial virus, or influenza virus in infants. Pediatrics 120(2):e410-e415.

18- Lindner J, Zehentmeier S, Franssila R, Barabas S, Schroeder J, Deml L, Modrow S (2008). CD4+

T helper cell responses against human bocavirus viral protein 2 viruslike particles in healthy adults. The Journal of infectious diseases 198(11):1677-1684.

19- Chung JY, Han TH, Kim JS, Kim SW, Park CG, Hwang ES (2008). Th1 and Th2 cytokine levels in nasopharyngeal aspirates from children with human bocavirus bronchiolitis. Journal of clinical virology 43(2), 223-225.

20- Call SA, Vollenweider MA, Hornung CA, Simel DL, McKinney WP (2005). Does this patient have influenza? Jama 293(8):987-997.

21- Wong-Chew RM, García-León ML, Noyola DE, Gonzalez LFP, Meza JG, Vilaseñor-Sierra A, Del Río- Almendarez CN (2017). Respiratory viruses detected in Mexican children younger than 5 years old with community-acquired pneumonia: a national multicenter study. International Journal of Infectious Diseases 62:32-38.

22- Kantola K, Hedman L, Tanner L, Simell V, Mäkinen M, Partanen J, Hyöty H (2015). B-cell responses to human bocaviruses 1–4: new insights from a childhood follow-up study. PLoS One 10(9):e0139096.

23- Ezzeldin N, Shalaby A, Saad-Hussein A, Ezzeldin H, El Lebedy D, Farouk H, Kandil DM (2012). Association of TNF-α–308G/A, SP-B 1580 C/T, IL-13–1055 C/T gene polymorphisms and latent adenoviral infection with chronic obstructive pulmonary disease in an Egyptian population. Archives of medical science: AMS 8(2):286-295.

24- Madhankumar AB, Mintz A, Debinski W (2002). Alanine- α-helix D segment of interleukin-13 reveals new functionally important residues of the cytokine. Journal of Biological Chemistry 277(45):43194-43205.

25- Vladich FD, Brazille SM, Stern D, Peck ML, Ghittoni R, Vercelli D (2005). IL-13 R130Q, a common variant associated with allergy and asthma, enhances effector mechanisms essential for human allergic inflammation. The Journal of clinical investigation 115(3): 747-754.

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13338 26- Nagashima H, Nakamura Y, Kanno H, Sawai T, Inoue H, Yamauchi K (2011). Effect of genetic

variation of IL-13 on airway remodeling in bronchial asthma. Allergology International 60(3):291-298.

27- Kumar A, Franssila R, Hedman K (2014). Immunobiology of Emerging Human DNAViruses.

28- Jartti T, Paul-Anttila M, Lehtinen P (2009). Systemic T-helper and T-regulatory cell type cytokine responses in rhinovirus vs. respiratory syncytial virus induced early wheezing: an observational study. Respiratory research 10 (1):85.

29- Mumtaz, M., & Hussain, N. . (2020). Rheumatoid Arthritis and the Role of VEGF Gene: An Overview. Journal of Scientific Research in Medical and Biological Sciences, 1(2), 75-90.

https://doi.org/10.47631/jsrmbs.v1i2.93

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