Human Adenovirus (HAdV) infection in Bangladesh

Abstract

Human adenoviruses (HAdVs) can cause wide spectrum of diseases. Overcrowding, malnutrition, and low socioeconomic condition are the predisposing factors for spread of virus in population. Two clinico-epidemiologically important diseases, diarrhea and ocular infections are present in our country. Type specific and group specific antibodies against adenoviruses show that Bangladeshi children are exposed to these viruses in their early life. Regrettably adenovirus research is not well practiced in our country in comparison of other viruses. Recently, identification of a novel human adenovirus type 65 (HAdV-D65) in Dhaka city signify the need molecular analysis of more samples from different areas in the country.

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Volume-2 Issue 1 July-2011
REVIEW ARTICLE
Human Adenovirus (HAdV) infection in Bangladesh
Prof. (Dr.) A K Adhikary1, Dr. Eiko Suzuki 2, Tsuguto Fujimoto3
1. Professor and Head, Department of Microbiology, BGC Trust Medical College, Chandanaish, Chittagong,
Bangladesh. Collaborative researcher, National Institute of Infectious Diseases (NIID), Tokyo, Japan.
2. Senior Researcher, Department of Developmental Medical Sciences, The University of Tokyo, Tokyo 162-860, Japan
3. Chief of Division 4, Infectious Disease Surveillance Centre, National Institute of Infectious Diseases (NIID), Tokyo,
Japan.
____________________________________________________________
Abstract:
Human adenoviruses (HAdVs) can cause wide spectrum of diseases. Overcrowding, malnutrition, and low
socioeconomic condition are the predisposing factors for spread of virus in population. Two clinico-
epidemiologically important diseases, diarrhea and ocular infections are present in our country. Type
specific and group specific antibodies against adenoviruses show that Bangladeshi children are exposed to
these viruses in their early life. Regrettably adenovirus research is not well practiced in our country in
comparison of other viruses. Recently, identification of a novel human adenovirus type 65 (HAdV-D65) in
Dhaka city signify the need molecular analysis of more samples from different areas in the country.
_____________________________________________________________
*Corresponding author
Professor (Dr.) Arun Kumar Adhikary
Mailing Address: BGC Trust Medical College
BGC Biddyanagar,Chandanaish,
Chittagong - 4000, Bangladesh.
E-mail: arun_adhikary@msn.com

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Volume-2 Issue 1 July-2011
Human adenoviruses (HAdVs) are non-
enveloped, double-stranded DNA viruses of the family
Adenoviridae, genus mastadenovirus (Figure 1)
Fifty-seven accepted types of human adenoviruses
(HAdVs) have been divided into seven species A
(HAdV-A) through G (HAdV-G) on the basis of
various biological and morphological criteria, nucleic
acid characteristics and homologies (deJong
1999; Benko, 2000; Jones, 2007) (Table 1). Nine
more proposed types including recently identified
HAdV-D65 from Dhaka city are waiting for approval.
Figure 1
Structure of human adenovirus. The icosahedral capsid
structure of adenovirus is made up of three proteins, hexon,
penton and fiber. A cross-sectional view of the viral capsid.
Some proteins are associated with viral DNA, whereas others
are associated with hexon and are involved in the formation
of the capsid.
Table. 1 Various species of human adenoviruses and their main tropism site
Species and cluster Adenovirus type Main tropism site
A 12, 18, 31 Gut
B:1 3, 7, 16, 21, 50 Respiratory tract and or eye
B:2 11, 14, 34, 35, 55 Urogenital system, respiratory tract (severe infection among
immunocompromised patient).
C 1, 2, 5, 6, 57 Respiratory tract (ARD among children) & gut
D 8–10, 13, 15, 17, 19, Eye (Epidemic keratoconjunctivitis)
20, 22–30, 32, 33,
36, 37-39, 42–49, 53, 54, 56
E 4 Respiratory tract
F 40, 41 Gut
G 52 Gut
The diagnosis of adenoviral infection directly from
clinical specimen is done by visualization of virus by
electron microscope (eg. stool specimen), and
demonstration of viral antigen by ELISA,
immunochromatography (ICT) or latex agglutination
test. Virus culture followed by neutralization test (NT)
is considered to be the “gold standard” for the
identification of HAdVs. These methods are
cumbersome, and majority of medical diagnostic
laboratories do not possess appropriate prototype
strain and serum collections. Moreover, 2 to 4 weeks
are required for the whole procedure. Therefore, now-
a-days gene based methods such as type specific
multiplex PCR, PCR-RFLP (restriction fragment
length polymorphism), and PCR with direct DNA
sequencing are in use. These methods are rapid,
sensitive and more specific than culture-NT (Adhikary
et al, 2001, Adhikary et al, 2004; Banik et al, 2005;
Takeuchi et al, 2001) (Figue 2).
HAdVs are the causative agent of a wide
spectrum of diseases such as, respiratory infections,
pharygoconjunctival fever (PCF), epidemic
keratoconjuctivitis (EKC), acute respiratory diseases
(ARD) in military recruits, gastroenteritis, acute
hemorrhagic cystitis, intussusception, and meningitis.
These viruses are also responsible for opportunistic
infections among the immunocompromised
individuals and transplant recipients resulting in high
mortality and morbidity (Berk, 2007). Among the
adenoviral diseases, gastroenteritis and ocular
infections are global public health problem.
Considering clinical and epidemiological
impact, in this review we have discussed about HAdV
research in Bangladesh , recent update, future prospect.
Adenoviral gastroenteritis: Gastroenteritis accounts
for approximately 1.76 million deaths of children
fewer than five years of age worldwide (Parashar,
2003). The viruses that cause gastroenteritis include
rotavirus, human adenoviurs, norovirus, astrovirus,
and sapovirus. HAdV-F40, HAdV-F41 & HAdV-G52
are known as enteric adenvoviruses (EAdV) because
they replicate efficiently in the intestine and are
excreted in the stool. HAdVs are the second most
agents after rotavirus, responsible for 1 to 20% of the
cases of viral diarrhea globally in both out patients and
hospitalized children (Murray, 1997). In Asia, 4.4% of
pediatric patients with diarrhea were shown to have
adenovirus-positive stool samples, with HAdV-F40
and 41 being the most prevalent types (Li et al, 2005).
Seroprevalence study of HAdVs in Dhaka,
Bangladesh
Detection of type specific antibodies against HAdVs
in a population is useful for epidemiological study. In
global level 50% of children less than 4 years of age
show presence of neutralizing antibody to EAdVs
(Kidd et al, 1983). In between February 1990 and May
1991, a study conducted by Jarecki-Khan and
Unicomb (1992) in Dhaka city revealed that 50% of

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Volume-2 Issue 1 July-2011
indoor patients between 0 and 24 months of age
showed antibodies to HAdV-C2, HAdV-F40, and HAdV-F41. IgG antibodies were more prevalent than
IgA antibodies.
Figure 2
Figure showing various conventional and molecular methods of detection and identification of human adenoviruses.
EM- electron microscopy, ICT-immunochromatography, EIA-enzyme immunoassay, HA- hemagglutination, HAI- hemagglutination
inhibition, NT- Neutralization test, PCR-polymerase chain reaction, RFLP-restriction fragment length polymorphism.
HAdVs as causative agents of diarrhea in Matlab,
Chandpur
With a view to uncover HAdVs as an important
causative agent of childhood diarrhea in Bangladesh,
4,409 stool specimens collected from under five
children seeking treatment for diarrhea in Matlab,
Bangladesh, from 1987 to 1991. The samples were
screened for the presence of HAdVs by EIA. EIA-
positive stool samples were typed with monoclonal
antibodies (MAb) specific for HAdV-F40, HAdV-41
and HAdV group antigen. HAdVs positive samples
were inoculated into Graham G293 cells, and retested
by EIA. Of adenovirus-positive cultures, 125 (2.8%)
specimens were confirmed as EAdVs, of which 51
(40.8%) were typed as Ad40 and 74 (59.2%) were
typed as Ad41, and 12 (0.3%) were identified as non-
EAdVs. In this study, HAdVs were responsible for
2.8%-12.3% diarrhea. The clinical features (Jarecki-
Khan, 1993) were significantly similar to symptoms of
infants infected with group A rotavirus such as watery
diarrhea (87.5%), more than eight loose bowel
movements per day in the 24-h period prior to
presentation (68.8%), with vomiting (80.0%o),
abdominal pain (76.3%), and low-grade fever
(95.0%o).
Molecular epidemiological study of HAdVs in
patients with diarrhea Dhaka City
In another study (Dey et al, 2009) 917 stool specimens
collected during the year 2004-2005 from infants and
children with acute diarrhea in Dhaka City, were
examined by molecular method in the University of
Tokyo. In multiplex PCR, 17 (1.9%) specimens were
positive for HAdVs. PCR and sequencing of hexon
gene showed that HAdV-F40, HAdV-D9 and HAdV-
D10 were responsible for 42% (7 of 17), 36% (6 of
17) and 22% (4 of 17) of cases, respectively.
Surprisingly, another important enteric adenovirus
HAdV-F41 was not detected. In this study, HAdVs
were most commonly observed in winter season
(October 2004 through January 2005) and in rainy
season (May 2005 through July 2005). This was the
first 1-year molecular epidemiological study of
HAdVs infection in Bangladesh.
Identification of novel human adenovirus type 65
(HAdV-D65) in Dhaka City
A novel recombinant HAdV designated as HAdV-D65
was discovered from the stool samples in Kawasaki
City Institute of Public Heath, Kawasaki, Japan. The
stool samples were originally collected during the year
2004-2005 from 4 children in Dhaka city who had
acute diarrhea. The virus was initially misidentified as
HAdV-D10 by hexon based PCR-sequence analysis
(Dey, 2009). However, whole genome sequence
revealed that HAdV-D10 is actually a recombinant
new adenovirus. HAdV-D65 is related to hexon gene
of HAdV-D10, penton base genes of HAdV-D37 and
HAdV-D58, and fiber gene of HAdV-D9.
(Matsushima et al, 2012).
Detec
t
ion
EM
ICT
EIA
Fiber len
g
th
Human Adenovirus
N
T
HAI tes
t
PCR Molecular t
yp
in
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PCR-enz
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me assa
y
Ph
y
lo
g
en
y
PCR-RFLP PCR-Microarra
y
S
p
ecies identification T
yp
in
g
HA tes
t
PCR

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Volume-2 Issue 1 July-2011
Ocular infection: HAdVs as agent of EKC in
Chittagong, Bangladesh
HAdVs cause outbreak as well as sporadic infection of
PCF and EKC. PCF is a relatively mild infection
characterized by conjunctivitis, pharyngitis, and fever
mostly among children and nearly all the patients
recover spontaneously. On the other hand, EKC is a
highly contagious and more serious ocular disease
characterized by severe bilateral conjunctivitis with
substantial corneal and extraocular involvement. After
an incubation period is 8 to 10 days a follicular
conjunctivitis with pain, edema of the eyelids,
lacrimation, and photophobia develop. Superficial
erosion of the cornea occasionally develops that can
ultimately cause corneal infiltration and opacity.
Corneal opacities in some cases can last for several
years or longer (Adhikary et al, 2001; Paval-Langston,
1994). The infection is transmitted through direct
contact with the infected area. In some patients, the
involvement slowly progress to a hemorrhagic
conjunctivitis that should be distinguished from the
rapidly evolving acute hemorrhagic conjunctivitis
associated with enterovirus 70.
Table. 2 Human adenovirus study in Bangladesh
References Disease Study place Sample Identification
Techniques Findings
Jarecki-Khan K, J
clin Microbiol
1992,30:2733-2734.
Healthy children
0-24 moths of age
Dhaka Serum ELISA Anti-HAdV-C2, Anti-
HAdV-4F0, Anti-HAdV-
F41
Jarecki-Khan K, J
clin Microbiol
1992;31:484–489.
Gastroenteritis
Less than 5 yrs of
age
Matlab Stool ELIIA,
Culture HAdV-F40 (40.8%),
HAdV-F41 (59.2%), NE-
HAdVs (0.3%)
Ohguchi T, ARVO
2003 Acute
conjunctivitis Chittagong Conjunctival
scrapings PCR-RFLP,
Culture, REA HAdV-B3, HAdV-E4,
HAdV-D8, HAdV-D8
(HAdV-8P, HAdV-8E)
Dey SK, Inf. Genet.
Evol. 2009;9:518–
522
Gastroenteritis
2-38 months of
age
Dhaka Stool PCR,
nucleotide
sequencing,
Phylogenetic
analysis
HAdV-D9 (36%),
HAdV-D10 (24%),
HAdV-F40 (40%)
Matsushima Y, Emer.
Inf. Dis. 2102;5 Gastroenteritis Dhaka Stool PCR,
nucleotide
sequencing,
Phylogenetic
analysis
HAdV-D65
Sixty six conjunctival swabs were collected from the
66 patients with suspected viral conjunctivitis in
BNSB Eye Infirmary, Chittagong from March to
September, 2002. ICT identified HAdVs in 11
samples. But 16 samples were positive in PCR
including the ICT positive 11 samples. RFLP of the
PCR product showed HAdV-D8, HAdV-B3, HAdV-
E4, HAdV-D37, and unclassified species B in 9, 2, 3,
1 and 1 sample, respectively. Restriction
endonucleases analysis (REA) of HAdV-D8 genome
reveals many genetic variants known as genome types.
Subsequent REA of HAdV-D8 isolates showed
genome type HAdV-8E in 5 samples, HAdV-8P in 3
and 1 was unclassified genome type. This study
elucidated HAdV-D8 as a major agent of EKC in
Bangladesh (Ohguchi et al, 2003).
Sporadic infection and periodic outbreak of EKC is
not uncommon in Asian countries including
Bangladesh. Outbreak in community, industry,
institution, and medical settings take place every year.
Nosocomial EKC often occurs, resulting in severe
outbreaks in Ophthalmology wards and necessitating
the restriction of clinical practices, such as the
postponement of eye surgery, the early release of
inpatients from hospital and the closure of
ophthalmology wards. Although not blinding, the
ocular infections create discomfort that leads to the
decreased quality of life and even possibly economic
damages (Adhikary, 2001). In Bangladesh, diagnosis
of viral conjunctivitis is done clinically and rapid
screening test (ICT) for HAdVs is not available in the
Ophthalmologist's office. Clinical presentations of
EKC may mimic chlamydial conjunctivitis, viral
conjunctivitis, allergic and bacterial conjunctivitis
(Adhikary et al, 2003). Thus, in medical setting, early
diagnosis of HAdV infection is undependable and
often flawed. This has been demonstrated by a 50%
rate of clinical inaccuracy compared to laboratory
confirmation (Leibowitz, 1976; Cheung, 2003). As the
therapeutic options depend on causative agent, correct
diagnosis is crucial for patient management and
prevention of disease spread.

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Volume-2 Issue 1 July-2011
Table 3 New adenovirus types reported from the year 2007
Type Source Country/Year
Recombination
(P H F) Reference
HAdV-G52 Stool USA/2003 P52H52F52 Jones et al, J virol 2007;81:5978-84
HAdV-D53 Conjunctival swab Germany/ 2006 P37H22F8 Walsh et al, Plos One
2009;4(6):e5635
HAdV-D54 Conjunctival swab Japan/2000 P54H54F54 Ishiko et al, J Clin Microbiol 2008;
46:2002-8
HAdV-B55 Nasopharygeal swab China/2006 P14H11+14F55 Walsh et al, J Clin Microbiol
2010;48:991-93
HAdV-D56 Pulmonary biopsy,
Conjunctival swab France/2008 P9H26F29 Robinson et al. Virology
2011;409:141-47
HAdV-C57 Nasopharygeal swab Russia/1997 H57F6 Walsh et al, J Clin Microbiol
2011;49:3482-90
HAdV-D58 Stool Argentina/1996 H58Freco
m
Liu et al. Plos One 2011; 6:e24491
HAdV-D59 Bronchoalveolar
Lavage (BAL) US/2007 P19H25F56 Liu et al. Plos One 2012; 7:e33212
HAdV-D60 NA Canada/2008 NA Robinson et al. (unpublished)
HAdV-A61 Stool Japan/2004 P31H6F31 Matsushima et al, J Gen Virol
2011;92:2770-75
HAdV-D62 AIDS patient UK/1993 NA Alissa et al. 2011 (unpublished)
HAdV-D63 NA USA/1959 P30H30F29 Singh et al. J Virol 2012;86(8):4693-
95
HAdV-D64 Carrel USA/1993 P22H19F37 Robinson et al (unpublished)
HAdV-D65 Stool Bangladesh/2005 P58H10F9 Matsushima et al, Emerg. Inf. Dis
2012;18: e
HAdV-B66 Nasophyayrngial
aspirate (NPA) Argentina/1987 P7H7F3 Dehghan et al. J Virol
2012;86(1):633-36
P- penton, H-hexon, F-fiber
NA- not available
In the last few years 14 new HAdVs (HAdV-G52 to
HAdV-D65) have emerged around the world pointing
out that the virus is evolving rapidly and most of them
are consequence of genetic recombination (Table 3).
This recombination event may be due to antiviral
immune pressure and co-infection with different
HAdV strains of the same species. The new viruses
can circumvent the existing immunity in human
population and create public health problems. Recent
detection of HAdV-D65 from Dhaka city implies the
possibility of finding more new adenoviruses in future.
Presence of neutralizing and group specific antibodies
against adenoviruses in Dhaka indicates that our
populations are exposed to adenovirus in their early
life. Circulations of major oculopathogenic stains of
HAdVs including HAdV-8 were observed in
Chittagong. On the other hand, two important
infections, adenoviral pneumonia among the children
and systemic infection within immunocompromised
individual (eg AIDS patient) are needed to be studied
in detail in our country. Regrettably, despite the
immense clinical importance, HAdV research in
Bangladesh is not well established. The local research
centers do not delve into this important virus even
though possessing molecular analysis facility.
Therefore, molecular analysis of adenoviral samples
from different places in Bangladesh could be helpful
to understand molecular epidemiology and
evolutionary tendency of the virus.

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Volume-2 Issue 1 July-2011
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