Searching for HAdV-52, the putative gastroenteritis-associated human adenovirus serotype in Southern Hungary.

Abstract

Human adenovirus (HAdV) serotype 52 has recently been discovered in the United States in samples from human patients with gastroenteritis of unknown etiology and is suspected to be a new human enteric pathogen. The aim of the present pilot study was to investigate whether this virus is circulating in the population of Southern Hungary by screening stool specimens collected from gastroenteritis cases and communal sewage samples in the area of Baranya County. A total of 209 diarrheic stool (124 from children and 85 from adults) and 45 influent sewage samples were screened for HAdV-52 by PCR using a primer pair specific to the gene of 12.5K protein in the E3 genomic region. The novel human adenovirus was not detected in any of the tested samples, suggesting that HAdV-52 was not circulating in the target population and the area during the study period. Since temporal and geographical fluctuations may markedly affect the epidemiology of human enteric pathogens, additional investigations are required to gain more in-depth insights into the ecology of this novel adenovirus.

Full text

Page 1

NEW MICROBIOLOGICA, 32, 185-188, 2009
Searching for HAdV-52, the putative
gastroenteritis-associated human adenovirus
serotype in Southern Hungary
Krisztián Bányai1,2,6, Vito Martella4, Edina Meleg1, Péter Kisfali3, Zoltán Péterfi5, Mária Benkö6,
Béla Melegh3, György Szu ”cs1,2
1Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pécs, Hungary;
2Department of Medical Microbiology and Immunology;
3Department of Medical Genetics and Child Development, Faculty of Medicine, University of Pécs, Pécs, Hungary;
4Department of Veterinary Public Health, University of Bari, Bari, Italy;
5Baranya County Hospital, Department of Infectology, Pécs, Hungary;
6Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary
Adenoviruses cause a variety of diseases in hu-
man, including lesions in the upper and lower
respiratory organs, the eye, the urinary tract and
the central nervous system, as well as hepatitis
and gastroenteritis (Horwitz, 2001).
Virtually all human adenovirus (HAdV)
serotypes have been found to be shed through
the feces and a variety of serotypes have been
detected in diarrheic stools. Thus far, however,
only two serotypes, HAdV-40 and HAdV-41
Corresponding author
Krisztián Bányai
Veterinary Medical Research Institute
Hungarian Academy of Sciences
Hungaria krt. 21, H-1143 Budapest, Hungary
E-mail: bkrota@hotmail.com
(members of virus species HAdV-F) have been
proven as a causative agent of acute gastroen-
teritis mostly in early childhood (Horwitz, 2001).
In 2007, a novel human HAdV serotype, HAdV-
52, was described from cases of diarrhea in
California, United States.
Based on phylogenetic analysis of homologous
genes and the overall gene content of the
genome, this virus has been proposed to consti-
tute a new species distinct from other well-char-
acterized species of HAdVs (Jones et al., 2007).
Because HAdV-52 shows significant homology
to some simian adenoviruses (SAdVs) isolated
from Old World monkeys, concerns have arisen
whether this new AdV serotype is an artifact,
generated by contamination of the primary
monkey cell culture used for the isolation of the
Human adenovirus (HAdV) serotype 52 has recently been discovered in the United States in samples from human pa-
tients with gastroenteritis of unknown etiology and is suspected to be a new human enteric pathogen. The aim of the
present pilot study was to investigate whether this virus is circulating in the population of Southern Hungary by
screening stool specimens collected from gastroenteritis cases and communal sewage samples in the area of Baranya
County. A total of 209 diarrheic stool (124 from children and 85 from adults) and 45 influent sewage samples were
screened for HAdV-52 by PCR using a primer pair specific to the gene of 12.5K protein in the E3 genomic region. The
novel human adenovirus was not detected in any of the tested samples, suggesting that HAdV-52 was not circulating
in the target population and the area during the study period. Since temporal and geographical fluctuations may
markedly affect the epidemiology of human enteric pathogens, additional investigations are required to gain more in-
depth insights into the ecology of this novel adenovirus.
KEY WORDS: Adenovirus, Diarrhea, Sewage, Hungary
SUMMARY
Received April 30, 2008
Accepted September 9, 2008

Page 2

virus. However, a second detection of the virus,
in an epidemiologically unrelated case of gas-
troenteritis, has suggested that HAdV-52 might
have already adapted to, and can perhaps be
transmitted in, the new host species (Jones et
al., 2007). These findings warrant further epi-
demiological surveys to assess the spread of this
novel AdV in various human populations, no-
tably in different geographical areas, and to es-
timate its role as a human enteric pathogen
more precisely.
We set up a pilot study in which a one-step PCR
assay was utilized using a primer pair specific to
HAdV-52 to test diarrheic stool specimens col-
lected from children and adults.
Nucleic acid extracts from influent sewage sam-
ples as surrogate of our epidemiologic survey
were also tested.
The PCR primers were designed to target the
gene coding for the 12.5K a protein within the
E3 genome region (Jones et al., 2007). The nu-
cleotide position of the forward (5` ATG ACA
GAT GGT GCG GCC GTG AGA GCT 3`) and re-
verse (5` TAA TCC GGG CGT GGG GCA GAT
GCA GAA C 3`) primers correspond to the ge-
nomic nt 25760 - 25786 and 26029 - 26002, re-
spectively, of serotype HAdV-52.
The choice of target was influenced by the fact
that although this gene is present in a variety of
non-enteric adenoviruses, it is absent in the
members of species HAdV-F (Davison et al.,
2003; Jones et al., 2007).
Such selectivity was an important prerequisite in
our study for screening sewage samples most of
which tested positive for enteric HAdVs. In sili-
co, the primers did not show a high ability to
cross react with a variety of DNA viruses known
to be shed in the human feces, and did not show
significant complementarities to other HAdV
and SAdV strains known to carry a homolog of
the 12.5K gene in their genome. The only ex-
ceptions were SAdV-1 and SAdV-7 that are ge-
netically closely related to HAdV-52 (Jones et al.,
2007).
The prototype strains of these viruses have been
isolated from Macaca fascicularis and Macaca
mulatta, respectively. Lacking a sample from
HAdV-52, we utilized SAdV-1 (strain ATCC VR-
195) and SAdV-7 (strain ATCC VR-201) for the
optimization of the reaction conditions, and al-
so as positive control in our assay.
The DNA from 10-20% suspensions of human
stool specimens was extracted by a standard
method using proteinase K digestion followed
by phenol:chloroform fractionation and then
ethanol precipitation.
Subsequently, the DNA was further purified with
the guanidine thiocyanate/silica method as de-
scribed by Boom et al. (1990). The nucleic acid
from raw sewage samples was extracted directly
by the same method as described in detail else-
where (Meleg et al., 2006, 2008). Five µl of sam-
ple DNA was added to the PCR mixture of 50 µl
final volume which contained 1X final concen-
tration of Taq polymerase buffer (75 mM Tris-HCl
(pH 8.8 at 25°C), 20 mM (NH4)2SO4, 0.01% (v/v)
Tween 20 and 2 mM MgCl2; Fermentas), 200 µM
each of the four dNTPs (Promega), 1 µM each of
the two primers (Integrated DNA Technologies),
and 2.5 U of Taq polymerase (Zenon). Cycling
conditions included an initial denaturation at
94°C for 5 min, followed by 40 cycles consisting
of steps at 94°C for 1 min, 60°C for 2 min, and
72°C for 1 min. The terminal elongation was at
72°C for 5 min. Fifteen µl PCR products were run
in 2% (w/v) agarose gel (Sigma) containing 0.5
µg/ml ethidium-bromide, and photographed us-
ing a UV transillumination.
A total of 124 diarrheic samples, collected from
children between 2004 and 2006, and 85 diar-
rheic stool samples from adults collected in 2006
in Baranya County, Hungary were screened.
Both sample sets had previously been tested on-
ly for group A rotaviruses by polyacrylamide gel
electrophoresis. With the exception of 6 adult
stool specimens, all samples tested negative for
rotavirus. In addition, 45 sewage samples col-
lected between 2004 and 2006 from the same
area as the clinical specimens were also
screened for HAdV-52. Both SAdV serotypes em-
ployed in our assay optimization procedure gave
amplicons of the expected size (270 bp), how-
ever no size specific amplicon was obtained with
any of the clinical or the environmental speci-
mens. In a few cases, nonspecific bands of size
similar to the expected one (~260 to 290 bp)
were obtained but the sequence analysis ruled
out their origin being from adenoviral DNA (da-
ta not shown).
A convincing amount of evidence supports the
theory of the supposed co-evolution and co-spe-
ciation of AdVs with their vertebrate host, in-
186
K. Bányai, V. Martella, E. Meleg, P. Kisfali, Z. Péterfi, M. Benkö, B. Melegh, G. Szu ”cs

Page 3

cluding rare examples for crossing the host
species barrier (Benkö and Harrach, 2003). In
some instances, however, host switching events
have been hypothesized to explain the striking-
ly close genetic relationships between AdV
serotypes derived from distantly related host
species. Such examples include a feline aden-
ovirus that is virtually indistinguishable from
HAdV-1 (from species HAdV-C) based on partial
nucleotide sequence of the hexon gene (Lakatos
et al., 1999), or the bovine AdV-9 that is closely
related to other serotypes of species HAdV-C
(Benkö et al., 1990).
A recent seroepidemiological study (Xiang et al.,
2006) demonstrated that 1.7% to 18.7% of indi-
viduals in cohorts of the African population in
the sub-Saharan area have antibodies to select-
ed AdV serotypes of chimpanzee origin, suggest-
ing that bush meat hunting may represent an oc-
cupational risk for acquisition of SAdV infection.
In contrast, zoo workers in the United States
who have frequent contact with chimpanzees
and thus are potentially exposed to chimpanzee
AdV infection are not at higher risk for infec-
tion than the control population, suggesting that
regular contact with chimpanzees is not a major
mode of transmission of heterologous aden-
oviruses (Xiang et al., 2006). All these findings
seem to be of interest and merit further scruti-
ny. Seroepidemiological studies with different
SAdV serotypes, concerning various contact pat-
terns between humans and monkeys or apes,
may shed light on the eventual zoonotic poten-
tial and the mode of transmission of heterolo-
gous adenoviruses.
It is currently unknown whether HAdV-52 needs
to be considered an emerging virus in the hu-
man population. At present, information on a
total of six human cases is available, five of
which came from a single gastroenteritis out-
break. A sixth case was independent from this
outbreak (Jones et al., 2007). This indicates that
the virus might have been in circulation for a
while in parts of the United States. As success-
ful in vitro propagation of HAdV-52 has been re-
ported (Jones et al., 2007), retrospective sero-
epidemiological surveys may be conducted to
better explore the distribution of this virus in
the general population in various geographic ar-
eas and to determine if antibody prevalence has
been increasing in a temporal fashion.
The present pilot study disclosed no evidence of
HAdV-52 infection in patients treated in hospi-
tal with acute gastroenteritis in a small area of
Hungary. Because communal sewage may serve
as an indicator for viral infections of low preva-
lence or unrecognized clinical importance of the
local population we sought to investigate the
presence of this virus in communal waste water
samples too.
Unlike conventional HAdVs (including both en-
teric and non-enteric serotypes) that were fre-
quently detected in these specimens (Meleg et
al., manuscript in preparation), no evidence was
found for shedding of HAdV-52 into the envi-
ronment.
In conclusion, the findings of this investigation
suggest that HAdV-52 was not circulating in the
surveyed area during the study period.
Evaluation of our results requires some caution.
Since temporal and geographical fluctuations in
the prevalence of enteric pathogens may have a
remarkable effect on the outcome of such epi-
demiologic surveys, additional investigations are
required to gain more data on the prevalence of
this novel adenovirus in the healthy population,
or in mildly or severely affected diarrheic pa-
tients.
ACKNOWLEDGEMENT
K.B. is a fellow of the Bolyai Janos Scholarship.
Part of this work was supported by grants (T
049020, K 67781) provided by the Hungarian
Scientific Research Fund (OTKA).
REFERENCES
BENKÖ, M., HARRACH, B., D’HALLUIN, J.C. (1990).
Molecular cloning and physical mapping of the
DNA of bovine adenovirus serotype 4: study of the
DNA homology among bovine, human and porcine
adenoviruses. J. Gen. Virol. 71, 465-469.
BENKÖ , M., HARRACH, B. (2003). Molecular evolution of
adenoviruses. Curr. Top. Microbiol. Immunol. 272,
3-35.
BOOM R., SOL C.J., SALIMANS M.M., JANSEN C.L.,
WERTHEIM-VAN DILLEN P.M., VAN DER NOORDAA J.
(1990). Rapid and simple method for purification
of nucleic acids. J. Clin. Microbiol. 28, 495-503.
DAVISON A.J., BENKO M., HARRACH B. (2003). Genetic
content and evolution of adenoviruses. J. Gen. Virol.
84, 2895-2908.
Searching for HAdV-52, the putative gastroenteritis-associated human adenovirus serotype in Southern Hungary
187

Page 4

HORWITZ M.S. (2001). Adenoviruses. In fields virology,
2301-2326. Edited by KNIPE D.M., HOWLEY P.M.
Philadelphia PA, USA. Lippincott Williams &
Wilkins.
JONES M.S., HARRACH B., GANAC R.D., GOZUM M.M.,
DELA CRUZ W.P., RIEDEL B., PAN C., DELWART E.L.,
SCHNURR D.P. (2007). New adenovirus species found
in a patient presenting with gastroenteritis. J. Virol.
81, 5978-5984.
LAKATOS B., FARKAS J., EGBERINK H.F., VENNEMA H.,
HORZINEK M.C., BENKÖ M. (1999). Detection of ade-
novirus hexon sequence in a cat by polymerase
chain reaction. Acta Vet. Hung. 47, 493-497.
MELEG E., JAKAB F., KOCSIS B., BÁNYAI K., MELEGH B.,
SZUCS G. (2006). Human astroviruses in raw sewage
samples in Hungary. J. Appl. Microbiol. 101, 1123-
1129.
MELEG E., BÁNYAI K., MARTELLA V., JIANG B., KOCSIS B.,
KISFALI P., MELEGH B., SZUCS G. (2008). Detection
and quantification of group C rotaviruses in com-
munal sewage. Appl. Environ. Microbiol. 74, 3394-
3399.
XIANG Z., LI Y., CUN A., YANG W., ELLENBERG S., SWITZER
W.M., KALISH M.L., ERTL H.C. (2006). Chimpanzee
adenovirus antibodies in humans, sub-Saharan
Africa. Emerg. Infect. Dis. 12, 1596-1599.
188
K. Bányai, V. Martella, E. Meleg, P. Kisfali, Z. Péterfi, M. Benkö, B. Melegh, G. Szu ”cs