Human Adenoviruses 40/41 and Cytokines Response in Children with Diarrhoea

Abstract

Background: One of the most significant etiologic factors in acute gastroenteritis in children is adenovirus types 40/41. Objective: The aim of this research was to diagnosis of adenovirus type 40/41 in children with diarrhoea in three Cities in Iraq; Babylon, AL-Najaf and Karbala, and evaluation of serum concentration of Interferon gamma (IFN-γ), Interleukin (IL) 6 and 10. Patients and methods: A total of 450 stool samples were collected from children with diarrhoea, age range from 1 to 36 month. Adenovirus 40/41 has been detected in stool using Polymerase Chain Reaction (PCR) method. Serum concentration of IFN-γ, IL-6 and 10 have been measurement using Enzyme-linked Immunosorbent Assay (ELISA) technique. Results: Out of 450 stool samples, there were 150 Children (33.3%) infected with adenovirus 40/41, the highest rate of infection was in the Karbala City with 73 infections (16.2%) followed by Najaf and Babylon were recorded 55 (12.2%) and 22 (4.8%) respectively. Cytokines profile revealed a significantly substantial rise in viral patients compared to controls. IFN-γ, IL-6 and IL-10 levels in the age range of 1 to 36 months were higher than those in the control group at 69.5, 61.3 and 112.3 pg/ml respectively. Conclusions: There were high infections with adenovirus type 40/41 in Karbala City. Interferon-γ, IL-6 and IL-10 play an important role in infection which can be used in immunotherapy.

Full text

The Egyptian Journal of Hospital Medicine (October 2022) Vol. 89 (2), Page 7025- 7030
7025
Received: 09/07/2022
Accepted: 14/09/2022
Human Adenoviruses 40/41 and Cytokines Response in Children with Diarrhoea
Layla Maki AL-Nasrawy1*, Sahar Mohammed Jawad2, Waleed Dakil AL-Nasrawy3
1 The General Directorate of Education Al-Najaf, Almutafawiqat Students School,iraq
2 Department of Physiology and Pharmacology, College of Nursing, Altoosi University College, Najaf, Iraq
3 Department of Biology, Faculty of Sciences, University of Kufa, Iraq
*Corresponding author: Layla Maki AL-Nasrawy, Mobile: 009647802424957, E-mail: alnasrawylaila@gmail.com
ABSTRACT
Background: One of the most significant etiologic factors in acute gastroenteritis in children is adenovirus types 40/41.
Objective: The aim of this research was to diagnosis of adenovirus type 40/41 in children with diarrhoea in three Cities
in Iraq; Babylon, AL-Najaf and Karbala, and evaluation of serum concentration of Interferon gamma (IFN-γ),
Interleukin (IL) 6 and 10. Patients and methods: A total of 450 stool samples were collected from children with
diarrhoea, age range from 1 to 36 month. Adenovirus 40/41 has been detected in stool using Polymerase Chain Reaction
(PCR) method. Serum concentration of IFN-γ, IL-6 and 10 have been measurement using Enzyme-linked
Immunosorbent Assay (ELISA) technique.
Results: Out of 450 stool samples, there were 150 Children (33.3%) infected with adenovirus 40/41, the highest rate of
infection was in the Karbala City with 73 infections (16.2%) followed by Najaf and Babylon were recorded 55 (12.2%)
and 22 (4.8%) respectively. Cytokines profile revealed a significantly substantial rise in viral patients compared to
controls. IFN-γ, IL-6 and IL-10 levels in the age range of 1 to 36 months were higher than those in the control group at
69.5, 61.3 and 112.3 pg/ml respectively.
Conclusions: There were high infections with adenovirus type 40/41 in Karbala City. Interferon-γ, IL-6 and IL-10 play
an important role in infection which can be used in immunotherapy.
Keywords: Adenovirus, 40/41, PCR, IFN-γ, IL-6, IL-10, case control study, University of Kufa.
INTRODUCTION
Adenovirus types 40 and 41, which cause 3.2 to
12.5% of all cases of acute diarrheal illnesses in children
and infants worldwide and have a higher detection rate
in underdeveloped nations, have been linked to acute
diarrheal diseases (1). Children under the age of two are
most commonly affected by adenovirus types 40 and 41,
which specifically infect the enterocytes of the small
intestine and produce chronic watery diarrhea that, if
ignored, can result in serious dehydration (2). In cell
culture, these viruses do not or do not grow well. By
virtue of their pathogen-associated molecular patterns
(PAMPs), such as capsid or DNA, adenovirus is well
known to activate innate immunity, which results in the
generation of IFN-γ and other cytokines such as IL-6
and IL-10 (3).
A cytokine with a variety of functions, IL-6 has
been demonstrated to be essential for the
immunological and host defense systems. In addition,
endogenous IL-6 has been demonstrated to work in
concert with other cytokines and growth factors to
prevent programmed cell death brought on by cytotoxic
drugs in viral infections (4,5).
One of the most powerful and promising
anticancer cytokines is IL-10. It is a heterodimeric
cytokine that is mostly generated by activated
macrophages and dendritic cells and is made up of two
distinct disulfide-linked subunits known as p35 and p40.
The production of interferon by natural killer cells, T
cells, dendritic cells, and macrophages is stimulated by
IL-12 (6,7). In the same way, it promotes T helper type 1
development and increases the cytolysis activity of
Natural killer (NK) cells and cytotoxic T lymphocytes
(CTLs) (8). Children and infants who live in areas with
poor healthcare access are particularly vulnerable to
diarrheal disease and the growth faltering, impaired
neurocognitive development, and increased mortality
are linked to repeated episodes of moderate-to-severe
diarrhoea and high rates of enteric pathogen infection
lead to defect in immune system due to increase
cytokines production (9,10).
Therefore, the aim of this research was to diagnosis
of adenovirus type 40/41 in children with diarrhoea in
three Cities in Iraq; Babylon, AL-Najaf and Karbala,
and evaluation of serum concentration of Interferon-γ
(IFN-γ), Interleukin (IL) 6 and IL-10.
PATIENTS AND METHODS
This case control study was performed in 3
cities; Babylon, AL-Najaf and Karbala during period
from 1st March 2021 to the end of August 2021. A total
of 450 stool samples were collected from children with
diarrhoea age range 1 to 36 month attending to the Al-
Jerahi Hospital (Babylon City), Al-Furat Al-Awsat
Hospital (AL-Najaf City) and AL-Zahra teaching
Hospital (Karbala City). In contrast, 60 healthy children
age range 1 to 36 month have been considered as control
group (11,12).
D i a g n o s i s o f a d e n o v i r u s :
Five gram of stool has been collected from each
children suffering from diarrhoea in disposable
containers (13,14). Monoplex PCR was done to detect
number of genes that encode for adenovirus type 40/41
to detect Penton base and Fiber genes (15,16) using
primers provided from (Bioneer Company /Korea)

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which are listed in Table 1 and amplification conditions
of genes were used by PCR reactions (Table 2).
Table (1): Types of primers that were used in PCR for detected adenovirus type 40/41.
PCR product
Sequence (5'→ 3')
Position
Gene
Primer
1,253bp
CTATCAGAACGACCACAGC
AACTT
TCCCGTGATCTGTGAGAGC
RG
14,152–
14,175
15,384–
15,404
Penton
base
Penton-F
Penton-R
1,153bp
TAATGTTTGTGTTACTCCG
CTC
GGGGAGGCAAAATAACTA
CTCG
31,180–
31,199
32,311–
32,332
Fiber
Fiber-F
Fiber-R
Table (2): Hexon gene and amplification conditions by PCR reactions.
Cytokines measurement:
Five ml of blood were collected from infected and
healthy children and left to clot at room temperature for
30 minutes to allow the clot to form, the clotted sample
were centrifuged at 1500 rpm for 20 minutes to obtain
serum and kept at deep freeze -20 ᵒC till used to
evaluation of concentration of IFN-γ, IL-6 and 10 using
ELISA technique (16,17). All kits provided from
Elabscience® ELISA Kit Germany.
Ethical considerations:
The study concept for human studies was
approved from Kufa University's College of Science
and AL-Kufa General Hospital by The Institutional
Ethics Committee. Additionally, before taking part
in the study, each individual gave written, informed
consent. This work has been carried out in
accordance with The Code of Ethics of the World
Medical Association (Declaration of Helsinki) for
studies involving humans.
Statistical analysis
Data was collected and analysed by using SPSS
(Statistical Package for Social Sciences, version 20,
IBM, and Armonk, New York). Quantitative data was
summarized as mean and standard deviation.
Qualitative data were given as number (n) and
percentage (%). Correlations were determined by
Spearman correlation. P-value <0.05 was considered
significant.
RESULTS
Total diarrheal infections:
Table 3 showed there were 450 children infected
with diarrhoea during period from 1st March 2021 to the
last August 2021. Age group 29-36 month was the
highest infected with 159 cases (35.3%) followed by
age group 1-8 month with 139 cases (30.9%). Most
infections were in Karbala City with 180 cases (40%)
followed by AL-Najaf City with 170 cases (37.7%) and
Babylon City with 100 (22.3%).
Table (3): Total diarrheal infections in children from 1st March 2021 to the last August 2021.
Age
City
1-8
9-18
19-28
29-36
Total (%)
Male
Female
Male
Female
Male
Female
Male
Female
Babylon
18
12
13
9
11
7
22
8
100 (22.3)
AL-Najaf
28
24
15
11
14
11
49
18
170 (37.7)
Karbala
33
24
17
16
16
12
39
23
180 (40)
Total (%)
139 (30.9)
81 (18)
71 (15.8)
159 (35.3)
450 (100)
Total adenovirus infections: Out of 450 stool samples have been tested with PCR technique, the results indicated that
there were 150 cases (33.33%) gave positive result with Penton base and fiber where were the sizes 1,253bp and 1,153bp,
respectively (Figures 1 and 2). Karbala City recorded 55 cases followed by AL-Najaf with 37 cases while, the lowest
infections were in Babylon City with 22 cases (Table 4).
Gene
Initial
denaturation
No. of
cycles
Denaturation
Annealing
Extension
Final
extension
Hexon
94 Cº for 5
min.
35
94 Cº for 1
min.
56Cº for 50 sec.
72 Cº for 50
sec.
72Cº for 1
min.

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Figure (1): PCR amplified products of Penton base gene of the adenovirus (Ad40 and Ad41) using specific primers
with expected size 1,253bp, DNA marker (100bp ladder).
Figure (2): PCR amplified products of fiber gene of the adenovirus (Ad40 and Ad41) using specific primers with
expected size 1,153bp, DNA marker (100bp ladder).
Table (4): Positive and negative infections in children with adenovirus from 1st March 2021 to the last August
2021
City
Total (%)
Total (%)
Positive infections
Negative infections
Babylon
22
78
100 (22.3)
AL-Najaf
37
97
170 (37.7)
Karbala
55
125
180 (40)
Total (%)
150 (33.33)
300 (66.67)
450 (100)
Serum concentration of cytokines: The results showed there was significant increase (P<0.05) in serum concentration
of IFN-γ, IL-6 and IL-10 as compared with control. All age groups of children infected with adenovirus showed
significant increase (P<0.05) as compared with control, and the high concentration detected in (29-36) age group
(69.5pg/ml) as compared with control (48.3pg/ml) in IFN-γ (Figure 3) and (61.3 pg/ml) compared to healthy control
group (35.3 pg/ml) in IL-6 (Figure 4) and (112.3 pg/ml) compared with healthy control group (70.5 pg/ml) in IL-10
(Figure 5). All correlation between cytokines and age groups has been mentioned in table 5.
Figure (3): IFN-γ level (pg/ml) in children with adenovirus and control.
Penton base gene
1,253bp
fiber gene
1,153bp

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Figure (4): IL-6 level (pg/ml) in children with adenovirus and control
Figure (5): IL-10 level (pg/ml) in children with adenovirus and control.
Table (5): Correlations between cytokines and age groups of children with adenovirus.
Correlation
Cytokine
Concentration
Adeno.
Age group
Cytokine
Pearson Correlation
1
-.031
.261
.000
Sig. (2-tailed)
.924
.413
1.000
N
12
12
12
12
Concentration
Pearson Correlation
-.031
1
.934**
.722**
Sig. (2-tailed)
.924
.000
.008
N
12
12
12
12
Adenovirus
Pearson Correlation
.261
.934**
1
.765**
Sig. (2-tailed)
.413
.000
.004
N
12
12
12
12
Age group
Pearson Correlation
.000
.722**
.765**
1
Sig. (2-tailed)
1.000
.008
.004
N
12
12
12
12
**Correlation is significant at the 0.01 level (2-tailed).

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DISCUSSION
In this study, the most children infected with
diarrhoea were in Karbala City with 180 cases (40%)
followed by AL-Najaf City with 170 cases (37.7%) and
Babylon City with 100 cases (22.3%). The risk of
diarrheal disease is especially high in children and
infants living in places where healthcare is inadequate
and growth falters, neurocognitive development is
impaired, and mortality is higher if repeated episodes of
moderate-to-severe diarrhoea occur. High rates of
enteric pathogen infections result in cytokines being
produced in excess, leading to immune system defects
(18,19). Polymerase Chain Reaction was used in this
study to diagnose adenoviruses by considering the many
advantages of this method. As we know, PCR is impor-
tant to diagnose viral DNA for high speed, sensitivity.
Adenoviral infections can also be diagnosed more
efficiently and effectively using PCR than commercial
kits. Only these two genes are detectable by primers
(Ad40 and Ad41). According to this study, out of 450
stool samples, it was found that enteric adenoviruses
caused only 150 cases (33.33%) of gastroenteritis in
children. It is higher than various studies such as Rezaei
et al.(20) and Kumthip et al. (21) where recorded (9% and
7.2%) respectively, it was evident in the present study
that types 40 and 41 adenovirus predominated. From
2003 to 2006, Banyai et al. (22) investigated diarrheic
patients of Hungry by PCR for adenovirus infection;
type Ad41 had been circulating all four years, but type
Ad40 had only been found in 2003 and 2004. The
incidence of adenovirus infection among gastroenteritis
patients has been attributable to both types 40 and 41 of
adenovirus, but recent investigations indicate that type
40 infection has declined and type Ad41 infection has
increased (23,24).
The current study concludes that the serum
concentrations of IFN-γ, IL-6 and IL-10 were
considerably higher in the children infected with
adenovirus than in the healthy control group. Strong
cellular immune responses, such as proliferative
responses and the release of Th1-type cytokines such as
IFN-γ following activation with certain viral antigens,
may be the cause of this adenovirus infections trigger
complicated immune reactions that involve both
humoral and cellular immune responses (25).
IFN-γ is known to increase the expression of
MHC class I and II on nucleated cells as well as
stimulate several effector actions in mononuclear
phagocytes. Its main purpose in living organisms seems
to be to stimulate macrophages to fight intracellular
infections like norovirus and adenovirus (26). Moreover,
several authors suggest that response to adenovirus
infections mainly involves IFN-γ secretion by CD4 Th1
T cells. Ponterio et al. (27) reported that patients
generate significant CD4 and CD8 IFN-γ responses to
specific adenovirus antigens during infection also these
observations suggest that an IFN-γ based detection
method could be used to diagnose individuals with
adenovirus infections.
In adenovirus infection, it has been shown that
IL-6 plays a central role in immune host defence, acute
phase reactions, and haematopoiesis in addition to
blocking programmed cell death induced by cytotoxic
agents and acting in cooperation with other growth
factors and cytokines, IL-6 plays a pleiotropic role. The
effect of IL-6 on cell-cell association, attachment, and
migration may also play a role in cell invasion and
metastases (5).
Thus, the role of IL-6 and IL-10 in several
malignancy pathophysiologies has clearly been
established and makes IL-6 a rationale target for
treatment of cancer explained increased pro
inflammatory cytokines IL-6 secretion with increased
adenovirus infection. In addition, IL-6 has been shown
to play an important role in several types of
malignancies (28).
Moreover, IL-10 has been shown to play an
important role in tumor-specific immunity. There is no
doubt that IL-6 is an essential cytokine for the immune
system and the host's defence system. In addition,
endogenous IL-6 has been shown to prevent
programmed cell death caused by cytotoxic drugs
during viral infections in conjunction with other
cytokines and growth factors (29). As a result of their
pathogen-associated molecular patterns, such as capsids
or DNA, adenoviruses activate the innate immune
system, which results in the release of IL-6 and IL-10
cytokines as well as IFN-γ.
In conclusion, there were high infections with
adenovirus type 40/41 in Karbala City. Interferon-γ, IL-
6 and IL-10 play an important role in infection which
can be used in immunotherapy.
Conflict of interest: There was no conflict of interest
in this study.
Funding: The funding by authors themselves
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