Advances in Infectious Diseases, 2013, 3, 257-262
Published Online December 2013 (http://www.scirp.org/journal/aid)
http://dx.doi.org/10.4236/aid.2013.34039
Open Access AID
257
Investigation of an Adenovirus-Induced Respiratory
Disease Outbreak
Xingyi Geng, Ji Zhang, Guoliang Yang
Jinan Municipal Center for Disease Control and Prevention, Jinan, China.
Received September 10th, 2013; revised October 9th, 2013; accepted October 16th, 2013
Copyright © 2013 Xingyi Geng, et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Objective: An epidemiological investigation was carried out in school X in Jinan, Shandong Province, China, to iden-
tify the cause, epidemiology, and etiological characteristics of a febrile respiratory disease outbreak; and therefore to
control the dissemination. Methods: Both field epidemiological investigations and laboratory examinations were car-
ried out. Results: Forty cases were identified, in which 38 cases were students and two were teachers. Clinical mani-
festations included fever, coughing, headache, and sore throat. A total of 21 pharyngeal swab specimens were collected
and 18 tested positive for adenovirus. The adenovirus hexon gene was sequenced in three of the 18 positive specimens
and the results showed a 100% homology with the standard HAdV-55 HEXO. Conclusions: The outbreak originated
from an adenovirus-infected student, who spread the pathogen to her classmates and teacher. The teacher then further
disseminated the disease within the school which led to 40 febrile respiratory infections.
Keywords: Epidemiology; Research Methods; Laboratory Examination; Adenovirus; Respiratory Disease
1. Background
Adenoviruses (AdV) are DNA viruses that typically
cause mild infections involving the upper or lower respi-
ratory tract, gastrointestinal tract, or conjunctiva [1]. Re-
placement of dominant serotypes by new strains usually
results in outbreaks in crowds. Outbreaks of AdV infec-
tion are more common in young children and military
recruits [2]. So far, more than 65 serotypes (subdivided
into groups A to F) of AdV have been identified by DNA
sequencing [3]. Of these, Ad-B55 was first isolated from
an acute respiratory disease outbreak in Shaanxi Province,
China in 2006. It is a re-emergent respiratory pathogen,
which was shown to be an intraspecies recombination be-
tween HAdV-11a and HAdV-14 [4,5]. Since then, there
have been no reported outbreaks worldwide.
In the present paper, we report an outbreak that in-
volved 38 students and two teachers at school X in Jinan,
Shandong Province, China. After an epidemiological in-
vestigation and DNA sequence homology, this outbreak
was shown to be caused by Ad-B55. As far as we know,
this is the second report on an outbreak of Ad-B55 in
schools in China.
2. Materials and Methods
2.1. Patients
Within a week, 40 teachers and students from school X
experienced at least one of following signs or symptoms:
unexplained fever (with a body temperature ≥38˚C), cough-
ing, sore throat, headache, throat congestion, swollen ton-
sils, purulent tonsils, or conjunctival hyperemia.
2.2. Epidemiological Investigation
An epidemiological investigation was carried out on the
40 reported cases to explore the possible cause and to
control the dissemination. Data including demographic
information, epidemiological characteristics, and clinical
manifestations were collected and studied for each case.
Throat swab specimens were sampled for some of the
cases and sent for laboratory examinations.
2.3. Laboratory Examinations
Throat swab specimens from 21 cases and serum speci-
mens from six acute cases were collected. Viruses were
isolated and cultured using two types of cell lines: Hep 2
and Vero cells. Nucleic acid was extracted using the Ro-
*Competing interests: The authors declare that they have no competing
interests.

Investigation of an Adenovirus-Induced Respiratory Disease Outbreak
258
che high-purity viral nucleic acid kit (Roche Applied
Science, Mannheim, Germany). Seven common respira-
tory tract pathogens were tested on 12 specimens using
RT-PCR (or PCR), including influenza virus (A\B) res-
piratory syncytial virus, parainfluenza virus, rhinovirus,
coronavirus AdV, and bocavirus. All 21 specimens were
tested for AdV using PCR. The sequencing results were
analysed using DNAStar and Seqman. The nucleotide
acid sequence homology was sequenced using MegAlign.
Mega5 was used for sequence alignment and phyloge-
netic tree construction.
2.4. Data Management and Statistics Analysis
Epi Data 3.0 was used to establish a database and SPSS
17.0 software was used for statistical analysis.
3. Results
3.1. School Background
The incident school is located in the northwest of Jinan
City, and covers an area of 14,700 m2 with a building
area of 4,025 m2. The teaching block has four floors:
grades 1 - 5 are located on levels one and two; grades 6 -
9 are located on levels three and four. There are two
stairs in the building; and each grade is relatively inde-
pendent. Teachers of each grade have separated offices.
The highest incidence of this outbreak was from class 1
and 2 of grade 8, which are located in the east wing of the
building (Figure 1).
There are 635 students (462 from grades 1 - 6, 173
from grades 7 - 9) and 72 members of staff in the school.
Students are distributed in 17 classes from 9 grades. It is a
non-boarding school with most of the students living in
nearby villages.
Some of the students from grades 7 - 9 participated in
the opening and closing ceremonies of a district sports
competition on March 27th and 28th 2012. It was the only
social gathering of students from the school close to the
time of the outbreak.
3.2. Clinical Manifestations and Epidemiological
Characteristics
3.2.1. Major Cli ni c al Mani festations
The main symptoms of the 40 reported cases included
fever (82.14%, highest reported body temperature 40˚C),
coughing and sore throat (78.57%), and headache
Figure 1. Floor plan of the teaching building.
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Investigation of an Adenovirus-Induced Respiratory Disease Outbreak 259
(64.29%). Some cases also experienced a runny nose, na-
sal congestion, sneezing, nausea, vomiting, and diarrhea.
Only one case developed pneumonia (Figure 2).
3.2.2. Time Distribution
The first case developed on March 17th. The number of
cases started to rise from the March 19th until April 1st;
a total of 40 people developed the disease (Figur e 3 ).
3.2.3. Populati on Distri bution
1) Occupation
There were 38 student infections (attack rate 5.98%) and
two infections among teachers (attack rate 2.78%).
2) Gender
There were 25 male cases and 15 female cases, with a
male to female ratio of 1.67:1.
3) Age
Most of the cases were aged 13 years old (n = 14) and 14
years old (n = 18), accounting for 35.0% and 45.0% of
the total incidence. Five cases were aged 15, accounting
for 12.5% of the total incidence.
4) Class distribution
The reported 38 student cases were mainly from class 1
and class 2 in grade 8. The attack rates of the two classes
were 52.78% for class 1 and 36.36% for class 2. Com-
pared to the total attack rate, the relative risk for these
two classes were 2.57 and 2.21 respectively. Class 2 in
grade 7 had the third highest attack rate, where four stu-
dents were infected. The attack rate for this class was
15.4%; other classes had below-average incidence rates
(Table 1).
3.2.4. Index Case Investigation
The index case was a 14 year-old female student from
class 1 in grade 8. On the night of March 17th, she began
coughing. Acute bronchitis syrup was self-administered
but without relief. The patient attended class the following
day while still coughing. On March 20th, she sought
medical assistance in the Dingzhuang outpatient clinic
and a five-day dose of anti-infection medication was pre-
scribed. On March 25th, she developed a fever with a body
temperature of 38.7˚C. On the same day, she was referred
to the General Military Hospital. Azithromycin and Co-
fetol cough syrup were prescribed, but the symptoms
persisted.
The patient was admitted to the Affiliated Hospital of
Shandong University of Traditional Chinese Medicine on
the morning of March 29th and underwent a CT scan. The
results suggested lower-right inflammation of the lung.
On the same afternoon the patient was referred back to the
Table 1. The grade distribution of 38 student cases.
Class Class
size
Number
of cases
Incidence
rate (%) RR 95%CI
Class 1 grade 836 19 52.78 2.57 1.69 - 3.91
Class 2 grade 833 12 36.36 2.21 0.93 - 5.23
Class 2 grade 726 4 15.38 0.75 0.29 - 1.93
Class 1 grade 654 2 3.70 0.18 0.04 - 0.72
Class 2 grade 336 1 2.78 0.14 0.02 - 0.95
Total 18538 20.54 REF
Note: two cases of teachers were not included in this analysis.
Figure 2. Main symptoms distribution of cases.
Figure 3. Cases onset time distribution.
Jinan Military Region General Hospital and was hospi-
talized. During the hospitalization period fever returned
with a body temperature of 39.9˚C.
1) Laboratory Diagnosis
A routine blood examination was carried out on March
30th. The results showed: white blood cell (WBC) counts
of 13.22 × 109, mononuclear cells 11.3%, a weak positive
test for mycoplasma pneumoniae serology, sputum cul-
ture growth of Streptococcus viridans, cardiac enzymes
(creatine kinase isoenzymes 30, myocardial ischemia
modified albumin 79.6, creatine kinase 374, α-hydroxy-
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Investigation of an Adenovirus-Induced Respiratory Disease Outbreak
260
butyrate the dehydrogenation acid 200). Another routine
blood examination was carried out on April 1st on the
index case, and the results showed that the WBC count
had dropped to 6.34 × 109 and the mononuclear count
was 11.2%.
2) Hospitalization
Treatment administered to the index case during hospi-
talization focused on anti-inflammation and symptom re-
lief. Medicines prescribed included 2.0 g ceftriaxone, 60
mg ambroxol hydrochloride intravenous infusion (once a
day). On March 31st the body temperature of the patient
began to fall and most symptoms improved; however, in
the morning diarrhea occurred, which lasted for one day
with a frequency of 4 - 5 times/day. By April 3rd, the pa-
tient was fully recovered and discharged from the hospi-
tal.
3.2.5. Key Case Investigation
The key case was a 35-year-old male teacher. He was the
Chinese teacher of the 8th grade as well as the class ad-
viser for class 2 in grade 8. He was responsible for five
lessons per class in grade 8 per week. On March 26th, he
started to develop fever (body temperature of 39.5˚C) as
well as body aches and tonsillar inflammation. He asked
for sick leave on March 27th and was given cephalospo-
rins and levofloxacin at a local clinic. The next day he re-
turned to school as his body temperature had dropped.
On March 29th, however, he had a recurrent fever and
headache; azithromycin and ribavirin were prescribed by
the clinic until the symptoms completely resolved.
The key case quarantined himself at home on the first
day that he started to develop symptoms (March 26th);
he also wore a mask and dined separately. His wife ex-
perienced an upper respiratory tract infection on the April
1st; other family members did not experience any symp-
toms.
4. Etiological Examinations
4.1. RT-PCR
Multiplex PCR was used to examine 12 of the 21 throat
swabs. The results were negative for influenza type A
and type B virus, respiratory syncytial virus, rhinovirus,
parainfluenza virus, coronavirus, and bocavirus nucleic
acid. All 21 throat swab specimens were tested for AdV
nucleic acid and 18 were found positive (positive rate of
85.71%).
4.2. Virus Isolation
All 21 throat swabs were isolated and cultured with Hep
2 and Vero cells. Two specimens were cytopathic (9.52%),
and PCR showed the presence of AdV.
4.3. AdV HEXON Gene Phylogenetic Analysis
Three positive specimens were sequenced for HEXON
and were named 2012-JN-357, 2012-JN-358, and 2012-
JN-362. The total length was 2841 bases. They were then
analyzed for nucleotide acid sequence homology with
MegAlign. Sequencing alignment was carried out and a
phylogenetic tree was built using Muscle of Mega5 (Fig-
ure 4). The three sequences in the gene evolutionary tree
located on the 55 branches of the AdV group B, were all
shown to have 100% homology with the AdV 55-type
control strain (GenBank Accession No. DQ874353.2).
4.4. Disseminating Factor Analysis
4.4.1. Time Analysis
The first infection occurred on March 17th, and the pa-
tient attended school until March 25th. The first genera-
tion of cases (n = 4) developed from March 17th - 22nd,
which were all from class 1 in grade 8. The second gen-
eration of cases (n = 36) developed from March 25th to
April 1st: 15 from class 1 in grade 8, 12 from class 2 in
grade 8, 4 from class 2 in grade 7, 2 from class 1 in grade
6, 1 from class 2 in grade 3, and two teachers.
4.4.2. Classro oms Location Analysi s
 Cases from class 1 in grade 8 were mostly sitting near
the first case.
 Class 2 in grade 8 was located next to class 1 in grade 8.
The same teacher was in charge of both classes, and he
was considered the source of infection.
 Both class 2 in grade 3 and class 2 in grade 7 were
located on the east wing of the building. Cases from
these two classes appeared during the late phase of the
outbreak. It is suspected that the shared stairs was the
route of transmission. Even though class 1 in grade 6
was located on the west wing of the building, all
cases from this class were infected on March 31st.
We suggest that they were infected by the already
spread outbreak in the school (Figures 1 and 5).
Figure 4. Complete HEXON gene sequence and AdV refer-
nce sequenc e gene phylogenetic tree. e
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Investigation of an Adenovirus-Induced Respiratory Disease Outbreak
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261
Figure 5. Eighth grade classes onset student seating distribution.
4.5. On-Site Control Measures
All reported cases were quarantined for observation and
treatment. Since a large number of patients were identi-
fied in a short period of time, it was recommended to the
school that it should have a seven-day holiday, combined
with the Ching Ming Festival public holiday. The advisor
teacher of each class should register any new incidence
and report to the Jinan municipal center for disease con-
troll and prevention in a timely manner. Classrooms
should be thoroughly ventilated before school starts
again and morning checks should be implemented. Edu-
cational flyers and notice boards should be used regard-
ing the outbreak as well as to eliminate unnecessary
panic.
5. Discussion
Based on the clinical manifestations of reported cases in
combination with laboratory examination results, this
outbreak was identified as an AdV-induced acute respi-
ratory disease outbreak. AdV infections mostly occur in
the winter and spring, and often cause outbreaks in the
military [6-9]. As the disease usually manifests a series
of common symptoms such as fever, sore throat, and
coughing; this presented difficulties in determining the
cause of the outbreak at an early stage [10]. With the evi-
dence provided by timely and accurate laboratory diag-
nosis, the outbreak was able to be identified within the
shortest time possible.
Respiratory infectious diseases normally spread rap-
idly and involve a large number of people. The transmis-
sion route is normally complex and the first case is often
difficult to identify. In the present study, after carrying
out a field investigation of the outbreak, it was deter-
mined that the first case was a 14 year-old female student.
Since students from different classes are relatively iso-
lated, we suggest the mutual teacher who was also in-
fected spread the disease across classes. Effective morn-
ing checks and registration of student absences are im-
portant measures for preventing the spread of the disease
within a school.
Human AdV can be divided into A ~G7 groups and 57
types based on their physical, chemical, and biological
properties. AdV infections induced by type 1, 2, 3, 5, and
6 AdV are more commonly seen, and the main symptoms
include fever, coughing, sore throat, and pneumonia; con-
junctivitis is seen in some cases [11]. AdV is one of the
main causes of acute respiratory infections among chil-
dren in China. Child acute laryngitis is mostly induced
by type 1, 2, 3, and 5 human AdV, and child atypical
pneumonia is mostly induced by type 1, 3, 4, and 7 hu-
man AdV. Pathogens for conjunctivitis are usually type 3,
7, and 14 human AdV; epidemic keratoconjunctivitis is
mostly caused by type 8 human AdV. Reports of AdV-
induced infant acute diarrhea have also been seen [12,13].
In recent years, type 55 AdV has been responsible for

Investigation of an Adenovirus-Induced Respiratory Disease Outbreak
262
multiple outbreaks in the military and in school. Due to
the lack of laboratory support in the past, many of the
outbreaks have been misidentified as influenza epidemics
[14]. With increasing use of nucleic acid testing techno-
logies, the ability to correctly recognise infectious patho-
gens has been substantially improved. Viruses tend to
survive longer in the winter and spring due to the low
temperature. In addition, students tend to conduct more
indoor activities during the winter and early spring, in a
relatively closed space with poor ventilation. Both fac-
tors contribute to viral respiratory disease transmission.
Consequently, winter and spring are peak seasons for
type 55 AdV infection to occur.
6. Conclusion
The outbreak originated from an adenovirus-infected stu-
dent, who spread the pathogen to her classmates and tea-
cher. The teacher then further disseminated the disease
within the school which led to 40 febrile respiratory in-
fections.
7. Acknowledgements
Thanks to Dr. Ji Zhang, for linguistic advice, Jinan Mu-
nicipal Center for Disease Control & Prevention.
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