Acta Med. Okayama, 2012
Vol. 66, No. 5, pp. 387ﾝ397
CopyrightⒸ 2012 by Okayama University Medical School.

http://escholarship.lib.okayama-u.ac.jp/amo/

Detection of Torque Teno Virus DNA in Exhaled Breath by
Polymerase Chain Reaction
Kumiko Chikasue, Miyuki Kimura, Kazuyuki Ikeda, Takuma Ohnishi,
Satoshi Kawanishi, Tomoe Iio, Mikio Kataoka, and Yujiro Arao＊‑

To determine whether exhaled breath contains(TTV) or not, we tested exhaled
breath condensate (EBC) samples by semi-nested PCR assay. We detected TTV DNA in 35ｵ (7/20) of
EBC samples collected from the mouth of one of the authors, demonstrating that TTV DNA isexcreted in exhaled breath with moderate frequency. TTV DNA was detected also in oral EBC samples from 4 of 6 other authors, indicating that TTV DNA excretion in exhaled breath is not an exception but rather a common phenomenon. Furthermore, the same assay could amplify TTV DNA fromroom air condensate (RAC) samples collected at distances of 20 and 40cm from a human face with 40(8/20) and 35ｵ (7/20) positive rates, respectively. TTV transmission has been reported to occur during infancy. These distances seem equivalent to that between an infant and its household members
while caring for the infant. Taken together, it seems that exhaled breath is one of the possible transmission routes of TTV. We also detected TTV DNA in 25ｵ (10/40) of RAC samples collected at a
distance of more than 180cm from any human face, suggesting the risk of airborne infection with
TTV in a room.
Key words:, exhaled breath, airborne infection, polymerase chain reaction(TTV) is one of human anelloviruses that have small non-enveloped virions
containing single-stranded, circular DNA genomes[1]. The more sensitive polymerase chain reaction(PCR) assays that have been recently developed havedemonstrated that 70 to 100ｵ of the general population throughout the world is chronically infected with
TTV except in the United Kingdom [2].
Although TTV has not yet been etiologically linked
to known diseases, TTV infection has been associatedwith accelerated disease development, poorer clinicalReceived December 13, 2012 ; accepted March 26, 2012.
Corresponding author. Phone :＋81ﾝ86ﾝ235ﾝ6893; Fax :＋81ﾝ86ﾝ235ﾝ6893
E-mail : yujiarao@md.okayama-u.ac.jp (Y. Arao)＊

outcome, higher morbidity and/or increased mortality
in many diseases [1]. For example, subjects withperiodontitis had a signiﬁcantly higher TTV DNA loadin the gingival tissue than those without disease [3].
TTV DNA prevalence and loads in both nasal swabs
and plasma were signiﬁcantly higher in patients withbronchopneumonia than in those with milder disease[4]. Serum prevalence of TTV DNA was signiﬁcantly higher in patients with severe idiopathic inﬂammatory myopathies than in those with milder disease[5].
Because there is neither an eﬀective antiviraltherapy nor a vaccine available for TTV at the present time, the only way to reduce the prevalence of
TTV and thereby counteract TTV-associated diseases

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Chikasue et al.

is to block TTV transmission from infected to uninfected individuals. Recently, Naganuma. [6]studied saliva samples from healthy children anddetected TTV DNA in 6, 34 and 90ｵ of children
aged 1, 4 and 42 months, respectively. From thisdata they concluded that TTV acquisition during theprenatal or neonatal period seemed infrequent and that
TTV prevalence increased with age and apparently
reached a plateau at or before 42 months of age.
Thus, we must prevent TTV transmission to children
less than 42 months old.
TTV DNA has been detected in various body ﬂuids
including semen [7, 8], cervical ﬂuid [9, 10], nasal
and throat secretions [4, 11], bile juice [12], breast
milk [13, 14] and saliva [14‑16]. The body ﬂuids ofparents and/or other household members have been
regarded as possible sources of TTV infection of theirchildren. If TTV is transmitted only via the body
ﬂuids, we can block TTV transmission to uninfectedchildren by protecting children from contact with
TTV-positive body ﬂuids. However, exhaled breathof the TTV-infected adults may be contaminated withthe virus since it passes through their nose, mouth andthroat, which are moistened by TTV-positive body
ﬂuids. If so, it would only be natural that the children
could be infected by TTV in the breath exhaled from
TTV-positive adults. In this case, we need to makeother plans to cope with TTV-contaminated exhaled
breath in addition to protecting children from contact
with TTV-positive body ﬂuids. However, there is no
information regarding contamination of exhaled breathwith TTV.
The aim of this study was to determine whether
exhaled breath had potential risk as a vehicle of TTVtransmission. In order to check for the existence of
TTV in exhaled breath, we collected exhaled breathcondensate (EBC) samples from the 7 authors bychilling exhaled air and examined the condensates forthe presence of TTV. Laboratory diagnosis of TTV
infection has been dependent on PCR detection of viral
DNA due to lack of other appropriate assays.
Unequivocal culture systems and well-characterizedanimal models are not available for the virus isolation.
There are no easy-to-use immunological methods todetect anti-TTV antibody and TTV antigens. Southernblot hybridization is not sensitive enough to detect
TTV DNA in samples such as body ﬂuids and serum.
In the present study, TTV presence in EBC samples

Acta Med. Okayama Vol. 66, No. 5

was tested by semi-nested PCR assay. As a result, wedemonstrated that TTV DNA was detectable innumerous EBC samples of 5 of the 7 authors.

Materials and Methods
We collected 20 samples oforal and nasal EBC and saliva from author A and 5
samples of oral EBC and saliva from each of authors
B, C, D, E, F and G. These seven authors included
4 males and 3 females. Their mean age (± standard
deviation) was 33.0 (± 13.0) years old and the age
range was 21‑53 years old. They were not suﬀeringfrom any illness during the sampling period. Also, wecollected 20 samples of room air condensate (RAC)
for each collection condition. Informed consent withappropriate documentation was obtained from ourselves. The EBC and saliva samples were collected
after gentle tooth brushing and mouth rinsing withwater. During the EBC collection, we kept normal
tidal breathing by nasal inhalation and oral or nasal
exhalation without coughing, sneezing or speaking.
Oral and nasal EBC and RAC samples were collectedusing 3 diﬀerent hand-made collection devices (Fig.
1).
Oral EBC samples were collected in the device
shown in the panel A of Fig. 1. The device was composed of a mouthpiece, a 10-ml pipette, a one-way
bulb and 2 bellows-tubes. The 2 bellows-tubes wereconnected by a one-way bulb. The mouthpiece was
attached to one end of the connected bellows-tubes andthe pipette was linked to another end. The bellowstube adjacent to the pipette was rotated to prevent
samples from contamination with saliva droplets. Allcomponents of the device were made of plastic and thedevice was used only one time. Exhaled air was sent
into the device via the mouthpiece, passed throughthe 2 bellows-tubes and one-way bulb and was chilledin the pipette by 2 refrigerants. The refrigerants werepre-cooled at −80℃. The ﬂuid that was accumulated
in the device for approximately 30min was poured into
a 20-ml plastic container and assayed as an oral EBC
sample.
Nasal EBC samples were collected by the same
method as oral EBC samples except for the collection
device (panel B of Fig. 1). The nasal EBC collection
device had a nosepiece and a straight bellows-tube
instead of the mouthpiece and rotated bellows-tube of

October 2012

Detection of TTV DNA in Exhaled Breath by PCRA

Refrigerant

PipetteExhaled air out

B

389

Bellows-tubes

One-way bulb

PipetteMouthpiece

Exhaled air infrom mouth

Bellows-tubes

Exhaled air out

Refrigerant

C

Polystylene tray

One-way bulb

Nosepiece

Exhaled air infrom nose

Exhaled air
from mouth
RefrigerantDistance from mouth to the polystylene tray was 20, 40, 80, 160 or more than 160 cm

Fig. 1
Schematic illustration of EBC and RAC collection devices. (A) Oral EBC collection device. Nasally inhaled air was exhaled into
the device through a mouthpiece held in a mouth, passed through a straight bellows-tube, a one-way bulb and a rotated bellows-tube thatprevented passage of saliva droplets. The breathed air that passed through the rotated bellows-tube was chilled in a pipette by pre-cooled
refrigerants at −80 ℃. The condensate that accumulated within both the pipette and the latter half of the rotated bellows-tube (the grayarea) was poured into a 20-ml plastic container and assayed as described in Materials and Method. (B) Nasal EBC collection device.
Nasally inhaled air was exhaled into the device trough a nosepiece inserted into nostrils, ﬂowed through 2 straight bellows-tubes and aone-way bulb between the tubes. Then, nasal EBC was trapped in the gray area, collected and assayed in the same way as the oral
EBC. (C) RAC collection device. Room air was cooled on the surface of a brand-new sheet of polyvinylidene chloride ﬁlm covering apolystyrene tray by the use of refrigerant on the opposite side of the tray. The refrigerant was pre-cooled at −80 ℃. RAC accumulated on
the ﬁlm (the gray area) was collected by a micropipette with a ﬁltered tip and assayed by the same methods as oral and nasal EBCs. RAC
was collected at distances of 20, 40, 80, 160 or more than 160 cm from a human face.

the oral EBC collection device. We inhaled nasally
through a hole in the top side of the nosepiece and sent
exhaled air into the device by closing the hole with agloved ﬁnger.
RAC samples were collected by an open systemusing the device shown in panel C of Fig. 1. This
device was made of a plastic tray (16 × 24 × 2cm),
the inner surface of which was covered with a brandnew polyvinylidene chloride ﬁlm. We breathed out
through the mouth and at a distance of 20, 40, 80,
160 or more than 160cm from the RAC collection
device. An RAC sample was accumulated on the surface of the ﬁlm for approximately 30min by chillingthe opposite side of the tray with refrigerant and wascollected into a 20-ml plastic container by a micropipette with a ﬁltered tip. The refrigerants were precooled at −80℃. The device was set at the sameheight as a human face and the collection side of thedevice fronted on the face when we collected RAC
samples at sites that were a speciﬁc distance (20 to
160cm) from a human face. Alternatively, when collecting at sites far from any face, the device wasplaced at a height of 180cm from the room ﬂoor, andthe collection side faced the room wall.
Saliva samples were collected by accumulating
saliva quietly in the oral cavity for approximately
10min and by drooling it into a 20ml plastic container.
There has been noreport about materials which are constantly containedin EBC and can be used as internal standards. For
example, the β-actin gene which has been used as aninternal control in many PCR experiments could not
be detected in 42ｵ of EBC samples [17]. Also, othermaterials such as a total DNA, which have been used
to standardize the amount of template DNA in PCR
assays, do not seem to be contained in EBC at the390

Chikasue et al.

same concentration. In order to make it possible todetect TTV DNA under the same condition without
using internal standards, we did not extract nucleic
acids from the samples and ampliﬁed TTV DNA fromexactly the same volume of sample solution. This assay
concept avoided the laborious use of an external standard to determine the extraction eﬃciency and also
was expected to reduce the risk of contamination of thesamples with exogenous target DNA.
To put the assay concept into practice, we pretreated the samples as follows: We froze each sample
at −80℃ immediately after collection and concentrated it by a Freeze dryer ALPHA 1‑2 (Martin
Christ Gefriertrocknungsanlagen Gm bH, Osterode
am Harz, Germany). After being thawed, the concentrated sample was supplemented with 10mM Tris•Cl(pH8.0), 1mM EDTA•2NA (pH8.0), 0.5ｵ SDS and
0.2mg/ml proteinase K and its volume adjusted to
one-tenth of that before the sample was concentrated
with DNase, RNase-free distilled water. Subsequently,
the sample was incubated at 53℃ for 3h to digest
proteins, further incubated at 95℃ for 10min to
inactivate the enzyme and then stored at −80℃ until
analysis by semi-nested PCR. For saliva, each sample
was supplemented with 10mM Tris•Cl (pH8.0), 1mM
EDTA•2NA (pH8.0), 0.5ｵ SDS and 0.2mg/ml proteinase K right after collection and treated in the same
way as EBC and RAC samples.
In addition to the samples for the semi-nested PCR
assay, EBC, RAC and saliva samples were stored at−80℃ for α-amylase assay without any pretreatment.
We detected TTV DNA in EBC, RAC andsaliva samples by the semi-nested PCR assay.
The primers for detection of TTV DNA were designed
based on already-reported TTV genome sequences. A
conserved region of the TTV genome was selected byaligning the genome sequences of 20 TTV strains(GenBank accession number AB008394, AB025946,
AB028668, AB028669, AB038619, AB038620,
AB064604, AB064605, AB064606, AB064607,
AF351132, AF435014, AJ620233, AJ620234,
AJ620235, AM712032, AM712033, AM712034,
AY666122 and NC002076), and 3 primers corresponding to the region were synthesized. The forward
and reverse primers of the outer pair were5ʼ-attttgctacgtcactaaccacg-3ʼ (TF5m) and 5ʼ-cccgaatt-

Acta Med. Okayama Vol. 66, No. 5gccccttgac-3ʼ (TR6), respectively. The forward andreverse primers of the inner pair were TF5m and5ʼ-taaactcacctccggcac-3ʼ (TR1c), respectively. TF5m,
TR6 and TR1c are located at nucleotide positions
1‑23, 204‑221 and 177‑194 in the TTV genome(AB008394), respectively. The predicted length of theamplicons produced by the inner primers is 194bp.
The ﬁrst PCR was performed in a 20- l reaction
mixture containing 1 × Taq DNA polymerase reaction
buﬀer (Roche Diagnostics K.K., Tokyo, Japan),
200 M of each deoxynucleotide triphosphate (Roche
Diagnostics K.K.), 0.5 U of Taq DNA polymerase(Roche Diagnostics K.K.), 0.1 M of forward andreverse primers, 10ｵ Tween 20 and 10 l of each
pretreated sample. In the cases of EBC and RAC,
10 l of the pretreated sample contained template
DNA quantitatively equivalent to that in 100 l ofeach sample before pretreatment. In the case ofsaliva, the amount of template DNA in a 10- l pretreated sample was equal to that in 9.18 l of each
untreated sample. The following ampliﬁcation program
was used for the ﬁrst PCR mixture: 7min at 94℃;
35 cycles of 30 sec at 94℃, 45 sec at 59℃ and 30
sec at 72℃; and a ﬁnal extension of 5min at 72℃.
Conditions for the second PCR mixture were the sameas those for the ﬁrst one except that forward andreverse primers were contained at a ﬁnal concentration of 1 M in place of 0.1 M; also, 1 l of the ﬁrst
PCR product was added instead of 10 l of pretreatedsample and Tween 20 was not included in the mixture.
Cycling conditions for the second PCR were the sameas the ﬁrst one except for the annealing at 63℃
instead of 59℃ and the cycling number of 25 times.
After ampliﬁcation was completed, aliquots of thesemi-nested PCR products were separated by electrophoresis (3ｵ agarose gels) and photographed under
UV light after ethidium bromide staining. The minimum detection limit of our semi-nested PCR wasdetermined using 10-fold serial dilutions of the cloned
TTV DNA fragment. It was estimated that 10 copiesof the fragment or more were detectable per reaction.
Negative controls for the PCR procedure weremade by adding water instead of sample solution, andno TTV DNA ampliﬁcation occurred after the second
PCRs. Positive controls for the PCR procedure wereprepared by adding water including 100 copies of thecloned TTV DNA fragment in place of the sample
solution, and we achieved the target DNA ampliﬁca-

Detection of TTV DNA in Exhaled Breath by PCR

Results
αFrom one of the present authors (authorA), we gathered saliva samples by drooling and collected oral and nasal EBC samples in the devices
shown in panels A and B of Fig. 1. Then, we examined the samples for activity of α-amylase, a wellknown enzyme that is secreted in saliva (Panels A, B
and C of Fig. 2). Detection of α-amylase activity in asample would serve as evidence that the sample contained saliva. While there were α-amylase activities
ranging from 107 to 505U/ml in saliva samples, theactivity of the enzyme was lower than the lowest
detection limit (0.0164U/ml) in all of the oral andnasal EBC samples. Thus, the oral and nasal EBC
samples seem not to have been contaminated withsaliva.

A

391

600
500
400
300
200
100
0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Saliva sample

α-amylase activty(U/ml)

B

600
500
400
300
200
100
0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

α-amylase activty(U/ml)

Oral EBC sample

C

600
500
400
300
200
100
0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Nasal EBC sampleD
α-amylase activty(U/ml)

tion after the second PCRs in all cases. The fragment
was cloned from the amplicon of the ﬁrst PCR. The
sequence identities between the fragment and each of
the above-described 20 TTV strains ranged from 76.8
to 94.1ｵ. In addition to controls for the PCR procedure, we collected 20 samples of outdoor air condensate as negative controls for sample collection andpretreatment. The outdoor air condensate samples
were collected on an emergency stairway of 5th ﬂoorof a building in the device used for collection of oral
EBC except that air was brought into the device by anelectric suction pomp. The samples were pretreatedand examined in the same way as described above, andall of the samples turned out to be negative for TTVDNA. The data of these controls are not shown in theﬁgures of this report.
αActivities of α-amylase in EBC, RAC and saliva samples were
measured enzymatically with Salivary α-Amylase
Assay Kit (Salmetrics LLC, PA, USA). Frozen
samples that were not pretreated were thawed, and8 l of each sample was assayed according to the
manufacturerʼs instruction.
The statistical signiﬁcance between sets of data was evaluated by thePearsonʼs chi square test with ＜ 0.05 being signiﬁcant. The relationship between sets of data was evaluated by calculating Pearsonʼs product-moment correlation coeﬃcient (r).

α-amylase activty(U/ml)

October 2012

600
500
400
300
200
100
0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
RAC sample

Fig. 2
Activities of α-amylase in saliva (A), oral EBC (B),nasal EBC (C) and RAC (D) samples. All saliva and EBC samples
were collected from author A as described in the Materials andMethods. RAC samples were collected at a distance of 20 cm fromthe face of author A. The lowest detection limit of the activity was
0.0164 U/ml.

Next, we tested the samples for the presence of TTV
DNA by semi-nested PCR assay. The assay ampliﬁed
TTV DNA from 85ｵ (17/20) of saliva samples(Panels A and D of Fig. 3), suggesting that author A
was surely positive for the virus and the EBC samples
from the author were worth being assayed for thevirus. Then, we assessed the EBC samples for thepresence of TTV DNA. We detected TTV DNA in
35ｵ (7/20) of oral EBC samples and in 10ｵ (2/20)
of nasal EBC samples (Panels B, C and D of Fig. 3).

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Acta Med. Okayama Vol. 66, No. 5A
300 bp 200 bp 100 bp -M 1 2 3 4 5

M 6 7 8 9 10 M 11 12 13 14 15 M 16 17 18 19 20

M 1 2 3 4 5

M 6 7 8 9 10

M 11 12 13 14 15

M 16 17 18 19 20

M 1 2 3 4 5

M 6 7 8 9 10

M 11 12 13 14 15

M 16 17 18 19 20

B
300 bp 200 bp 100 bp -C
300 bp 200 bp 100 bp -TTV positivity (%)

D

100
80
60
＊

40
20
0

＊
SalivaOral EBC

Nasal EBC

SampleFig. 3Detection of TTV DNA in exhaled air. Electrophoretic proﬁles of the semi-nested PCR products ampliﬁed from saliva, oral EBC
and nasal EBC samples are shown in panels A, B and C, respectively. We collected all samples from author A and assayed them as
described in Materials and Methods. Signals observed at a position of approximately 200 bp were positive signs for TTV DNA. The abbreviation M means a lane of molecular markers. (D) TTV DNA-positive rates in saliva, oral EBC and nasal EBC samples. Columns represent
the TTV DNA-positive rates calculated from the results in the panels A, B and C. A single asterisk means that the value is signiﬁcantly
lower than that in saliva samples ( ＜ 0.05).

The results indicate that exhaled breath from theauthor A undoubtedly contained TTV DNA, although
the TTV DNA-positive rates in the oral and nasal
EBC samples were signiﬁcantly lower than that of thesaliva samples.
To determine whether or not author Aʼs
exhalation of TTV DNA was an exception, we examined six other authors (authors B, C, D, E, F and G)
using semi-nested PCR. With regard to saliva, wedetected TTV DNA in 90ｵ (27/30) of the samples
and found that all of the 6 authors were positive forTTV (Panels A and C of Fig. 4). As the result of
examining oral EBC samples from these TTV-positive
authors, we could detect TTV DNA in 20ｵ (6/30) of
the samples and in 4 of the 6 authors (Panels B and C
of Fig. 4). Therefore, it seems that excretion of TTV
in exhaled breath is not an exceptional case but iscommon.
Although the above-described TTV
DNA-positive data demonstrated TTV DNA excretionin exhaled breath, the data were obtained by closed
systems using devices in which the exhaled breath wasOctober 2012

Detection of TTV DNA in Exhaled Breath by PCRA
300 bp -

M

1

2

3

4

5

M

6

7

8

9

10

393

M 11 12 13 14 15

200 bp 100 bp 300 bp -M 16 17 18 19 20

M 21 22 23 24 25 M 26 27 28 29 30

MM

200 bp -100 bp -B
300 bp -

1

2

3

4

5

10

M 11 12 13 14 15

M 21 22 23 24 25

M 26 27 28 29 30

6

7

8

9

200 bp -100 bp 300 bp -M 16 17 18 19 20

200 bp 100 bp -TTV positivity (%)

C

100
80
60
40
＊

20
0

SalivaSampleOral EBC

Fig. 4Generality of TTV DNA detection in exhaled breath. Electrophoretic proﬁles of the semi-nested PCR products ampliﬁed fromsaliva and oral EBC samples are shown in panels A and B, respectively. We collected and assayed saliva and oral EBC samples in thesame way as in Fig. 3 except for the authors from whom the samples were collected. Samples 1 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to
25 and 26 to 30 were collected from authors B, C, D, E, F and G, respectively. Signals observed at a position of approximately 200 bp
are positive signs for TTV DNA. The abbreviation M means a lane of molecular markers. (C) TTV DNA-positive rates in saliva and oral
EBC samples. Columns represent the TTV DNA-positive rates calculated from the results in panels A and B. A single asterisk means thatthe value is signiﬁcantly lower than that in the saliva samples ( ＜ 0.05).

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Acta Med. Okayama Vol. 66, No. 5

not diﬀused (Panels A and B of Fig. 1). In actual
circumstances, there is no tube that connects an
excretor of TTV directly to a recipient. Thus, it wasnecessary to verify whether or not the room air neara human face contains TTV. We collected RAC samples at distances of 20, 40, 80 and 160cm from theA
300 bp 200 bp 100 bp -B
300 bp 200 bp -face of author A using the device shown in panel C ofFig. 1. We quantiﬁed α-amylase activity in 20 RAC
samples collected at a distance of 20cm from the facebut could not detect any activity of the enzyme in thesamples (Panel D of Fig. 2), suggesting that the RAC
samples were not contaminated with saliva. Then, we

M 1 2 3 4 5

M 6 7 8 9 10

M 11 12 13 14 15

M 16 17 18 19 20

M 1 2 3 4 5

M 6 7 8 9 10

M 11 12 13 14 15

M 16 17 18 19 20

M 1 2 3 4 5

M 6 7 8 9 10

M 11 12 13 14 15

M 16 17 18 19 20

M 1 2 3 4 5

M 6 7 8 9 10

M 11 12 13 14 15

M 16 17 18 19 20

100 bp -C
300 bp 200 bp 100 bp -D
300 bp 200 bp 100 bp -TTV positivity (%)

E

50
40

＊

＊＊

30
20
10
0

20

40
80
160
A distance from the human face (cm)Fig. 5Detection of TTV DNA in room air near a human face. Electrophoretic proﬁles of the semi-nested PCR products ampliﬁed from
RAC samples obtained at distances of 20, 40, 80 and 160 cm from the face of the author A are shown in panels A, B, C and D, respectively. We collected and assayed RAC samples as described in Materials and Methods at the same height as the face of the author A.
Signals observed at a position of approximately 200 bp are positive signs for TTV DNA. The abbreviation M means a lane of molecularmarkers. (E) TTV DNA-positive rates in RAC samples obtained at distances of 20, 40, 80 and 160 cm from the face. Columns represent
the TTV DNA-positive rates calculated from the results in panels A, B, C and D. A single asterisk means that the value is signiﬁcantly
higher than those of RAC samples at distances of 80 and 160 cm from the face, and a double asterisk means that the value is signiﬁcantly
higher than that in RAC samples at a distance 160 cm from the face ( ＜ 0.05).

October 2012

Detection of TTV DNA in Exhaled Breath by PCRscreened the RAC samples for presence of TTV DNA
by the same methods as described above (Fig. 5).
As a result, we found that TTV DNA was detectable in RAC samples collected at distances of 20 and
40cm from a human face with positive rates of 40(8/20) and 35ｵ (7/20), respectively. These TTV
DNA-positive rates were comparable to that in oral
EBC samples from the same author. Thus, it seems
that room air near a human face contains an equivalentamount of TTV to orally exhaled breath, and thatexhaled breath may be one of the possible transmission routes of TTV in an actual situation. We
detected TTV DNA also in RAC samples collected at
distances of 80 and 160cm from the face. There wasA
300 bp 200 bp -395

a negative correlation between the TTV DNA-positive
rates and the distances from the face (r ＝ −0.90).
The correlation seems to indicate the natural diﬀusionof TTV in room air.
We could detect TTV DNA in
RAC samples that were collected not only at sites
near a human face (20 and 40cm) but also at a site far
from the face (160cm). This result may suggest that
TTV can drift in room air for a long distance. To
better understand the distribution of TTV in room air,
we collected RAC samples at 2 diﬀerent room sites
located at a height more than 180cm from a room ﬂoor
and at a distance more than 160cm from any human

M 1 2 3 4 5

M 6 7 8 9 10

M 11 12 13 14 15

M 16 17 18 19 20

M 1 2 3 4 5

M 6 7 8 9 10

M 11 12 13 14 15

M 16 17 18 19 20

100 bp -B
300 bp 200 bp 100 bp -TTV positivity (%)

C

50
40

＊

30
20
10
0

Dead end

Entrance
A position in the room

Fig. 6Detection of TTV DNA in room air far from a human face. Electrophoretic proﬁles of the semi-nested PCR products ampliﬁed
from RAC samples obtained at the dead end and entrance of the room are shown in panels A and B, respectively. We collected RAC
samples in the same way as in Fig. 5 except that the samples were collected at a height of 180 cm from the room ﬂoor and at distances ofmore than 160 cm from any human face and assayed in the same way as Fig. 3. Signals observed at a position of approximately 200 bp
are positive signs for TTV DNA. The abbreviation M means a lane of molecular markers. (C) TTV DNA-positive rates in RAC samples
obtained at the dead end and entrance of the room. Columns represent the TTV DNA-positive rates calculated from the results in panels
A and B. The single asterisk means that the value is signiﬁcantly higher than that in RAC samples collected at the entrance of the room
( ＜ 0.05).

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Acta Med. Okayama Vol. 66, No. 5

face. The collection side of the device used to accumulate RAC did not face any human in the room but
faced a room wall. We examined the RAC samples forthe presence of TTV DNA by the semi-nested PCR
assay, resulting in ampliﬁcation of TTV DNA fromthe samples with a much higher frequency than we had
anticipated (Fig. 6). TTV DNA was detected in 40ｵ(8/20) of the samples that were collected at the deadend of the room and in 10ｵ (2/20) of the samples thatwere collected at the entrance of the room. The result
suggests a wide distribution of TTV in room air. The
diﬀerence in the TTV DNA-positive rate between thedead end and entrance of the room was statisticallysigniﬁcant, suggesting that the virus may accumulate
in the inner part of the room.

Discussion
First, we demonstrated TTV DNA in both oral andnasal EBC samples from author A. The mean TTV
DNA-positive rate in oral EBC samples reached 35ｵ,
indicating that the presence of TTV DNA is not rare
in exhaled air. Second, we detected TTV DNA in oral
EBC samples not only from author A but also from 4
of 6 other authors examined in the following investigation, suggesting that excretion of TTV in orally
exhaled air is not unique to author A but is a common
phenomenon. Third, we detected TTV DNA also in
RAC samples collected at distances of 20 and 40cm
from a human face with positive rates of 40 and 35ｵ,
respectively. These TTV DNA-positive rates arecomparable to that of oral EBC samples. Thus, it
seems that exhaled air contains TTV in actual situations. TTV transmission occurs during infancy [6]and it has been assumed that saliva droplets fromhousehold members of an infant are the sources of
TTV transmission to the infant. The above-described
results suggest that exhaled air from household members may be a possible source of TTV transmission to
the infant in addition to saliva droplets.
Although α-amylase activity was more than 107U/
ml in saliva, the activity of the enzyme in exhaled air
was below the lowest detection limit (0.0164U/ml).
Thus, it seems that our oral EBC samples were notcontaminated with saliva, and the TTV DNA-positive
results obtained in the oral EBC samples were notdirectly due to the contamination of samples with the
TTV DNA-positive saliva. Nevertheless, the TTV

DNA-positive rate in saliva samples from author A wassigniﬁcantly higher than those in oral and nasal EBC
samples collected from him. Hence, we can not completely deny the possibility that TTV in EBC samples
is associated with the virus secreted in saliva; theorigin of TTV DNA in the EBC samples is still
unknown.
In addition to directly exhaled breath and room airnear a human face, we could detect TTV DNA also inroom air far from human faces, suggesting that TTV
drifts for long distances and is widely distributed inroom air. During RAC sample collection at sites far
from a human face, the ventilation of the room was not
so good because the air in the room was ventilated
only through the entrance door and the door was
opened for a short time, when someone entered orexited the room. Since TTV is a non-enveloped virus,
TTV is considered highly resistant to environmental
stressors [18]. For example, TTV infectivity has
been reported not to be lost even after 95h of dry heat
treatment [19]. Together with this remarkable stability of TTV particles, our result may suggest therisk of airborne infection with TTV in rooms without
good ventilation.
Breath is exhaled constantly by TTV-infected persons, and so the generation frequency of exhaled
breath is much higher than those of other vehicles of
TTV spread. TTV acquisition seems to occur during
infancy in most cases [6]. It is assumed that membersof a household in which there is an infant may attempt
to protect the infant from contact with their body ﬂuids but not usually from their exhaled air. As for therisk of TTV transmission, therefore, it seems possible that airborne infection via exhaled air may becomparable to infection though other routes. However,at this moment in time, no one can evaluate the risk ofairborne infection with TTV quantitatively as we cannot estimate TTV infection eﬃciencies in humans via
other vehicles of spread, largely because there is a
paucity of experimental systems to determine TTV
infection eﬃciency. Infection experiments in humans
cannot be performed for ethical reasons. Also, practical animal models for the quantitative assay of TTV
infection are not available. In addition, studies of theinfectivity of TTV are very limited due to lack of celllines that support suﬃcient TTV replication. Thesediﬃculties should be resolved in order to enable risk
assessment of TTV transmission routes includingOctober 2012

Detection of TTV DNA in Exhaled Breath by PCR

airborne infection.
Even in oral EBC from the same author, there
were both TTV DNA-positive and -negative samples.
This suggests the possibility that diﬀerences in thephysical and mental conditions of a host in ordinary
life may inﬂuence TTV replication and/or TTV
excretion from the host. Information on the conditionsaﬀecting TTV replication and excretion would seem
useful for preventing transmission of the virus andrelated disease deterioration.

8.

9.
10.
11.
12.

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