Roden s and Risk in he Mekong Del a o Vie nam:
Se op e alence o Selec ed Zoono ic Vi uses
in Roden s and Humans
Nguyen Van Cuong,
1
Juan Ca ique-Mas,
1
Hien Vo Be,
2
Nguyen Ngoc An,
3
Ngo T i Tue,
1
Nguye Lam Anh,
1
Pham Hong Anh,
1
Nguyen The Phuc,
1
S ephen Bake ,
1
Liina Vou ilainen,
4
Anne Ja¨a¨skela¨inen,
5,6
Eili Huh amo,
5
Mi a U iainen,
5
Ta ja Si onen,
5
An i Vahe i,
5,6
Heikki Hen onen,
4
Olli Vapalah i,
5–7
Yannick Cha al,
8
Se ge Mo and,
8
and Julie E. B yan
1,9
Abs ac
In he Mekong Del a in sou he n Vie nam, a s a e commonly aded in we ma ke s and sold li e o ood
consump ion. We in es iga ed se op e alence o selec ed g oups o oden -bo ne i uses among human popu-
la ions wi h high le els o animal exposu e and among co-loca ed oden popula ions. The indi ec luo escence
an ibody es (IFAT) was used o de e mine se oposi i i y o ep esen a i e e e ence s ains o han a i uses
(Dob a a i us [DOBV], Seoul i us [SEOV]), cowpox i us, a ena i uses (lymphocy ic cho iomeningi is i us
[LCMV]), la i i uses ( ick-bo ne encephali is i us [TBEV]), and oden pa echo i uses (Ljungan i us), using
se a om 245 humans li ing in Dong Thap P o ince and 275 oden s ep esen ing he i e common oden species
sold in we ma ke s and p esen in pe idomes ic and a m se ings. Combined se oposi i i y o DOBV and SEOV
among he oden s and humans was 6.9% (19/275) and 3.7% (9/245), espec i ely; 1.1% (3/275) and 4.5% (11/
245) o cowpox i us; 5.4% (15/275) and 47.3% (116/245) o TBEV; and exposu e o Ljungan i us was 18.8%
(46/245) in humans, bu 0% in oden s. Ve y li le se o eac i i y was obse ed o LCMV in ei he oden s (1/275,
0.4%) o humans (2/245, 0.8%). Molecula sc eening o oden li e issues using consensus p ime s o la i-
i uses did no yield any amplicons, whe eas molecula sc eening o oden lung issues o han a i us yielded one
han a i us sequence (SEOV). In summa y, hese esul s indica e low o mode a e le els o endemic han a i us
ci cula ion, possible ci cula ion o a la i i us in oden ese oi s, and he i s a ailable da a on human exposu es
o pa echo i uses in Vie nam. Al hough he cu en e idence sugges s only limi ed exposu e o humans o known
oden -bo ne diseases, u he esea ch is wa an ed o assess public heal h implica ions o he oden ade.
Key Wo ds:Vie nam—Roden s—Fla i i us—Han a i us—Cowpox i us–A ena i us—Ljungan i us.
In oduc ion
In he Mekong Del a o Vie nam, a s a e commonly
apped in ice ields be o e and a e ha es ing and
sold li e o ood consump ion. The a ade is igo ous,
in ol ing up o 3300–3600 onnes o mea sold pe yea
(Khiem e al. 2003). Ra in es a ions in ice ields cause
eno mous c op damage, and apping p o ides a means o
pes con ol as well as augmen ing local die a y sou ces o
animal p o ein. Roden pa s (heads, ails, and inna ds) a e
also o en ed o domes ic li es ock o ep iles ea ed in ca-
pi i i y ( ogs, snakes, c ocodiles). Roden s a e well- ecognized
1
Ox o d Uni e si y Clinical Resea ch Uni , Hospi al o T opical Diseases, Ho Chi Minh Ci y, Vie nam.
2
Sub-Depa men o Animal Heal h and
3
Depa men o P e en i e Medicine, Dong Thap P o ince, Cao Lanh, Vie nam.
4
Finnish Fo es Resea ch Ins i u e, Van aa, Finland.
5
Haa man Ins i u e, Uni e si y o Helsinki, Helsinki, Finland.
6
HUSLAB, Depa men o Vi ology and Immunology, Helsinki Uni e si y Cen al Hospi al, Helsinki, Finland.
7
Facul y o Ve e ina y Medicine, Depa men o Ve e ina y Biosciences, Uni e si y o Helsinki, Helsinki, Finland.
8
Uni e si e
´Mon pellie , Mon pellie , F ance.
9
Nu ield Depa men o Medicine, Ox o d Uni e si y, Ox o d, Uni ed Kingdom.
ªNguyen Van Cuong e al. 2015; Published by Ma y Ann Liebe , Inc. This Open Access a icle is dis ibu ed unde he e ms o he
C ea i e Commons License (h p://c ea i ecommons.o g/licenses/by/4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in
any medium, p o ided he o iginal wo k is p ope ly c edi ed.
VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 15, Numbe 1, 2015
Ma y Ann Liebe , Inc.
DOI: 10.1089/ bz.2014.1603
65
ec o s o a ange o zoono ic i uses ansmi ed ia close
con ac wi h sali a, u ine, o eces (Mee bu g 2010).
Gi en he equency and in ensi y o human and li es ock
con ac wi h oden s, we aimed o explo e po en ial isks
associa ed wi h hese exposu es h ough se op e elance
s udies. We de e mined se op e alence o i e g oups o
known zoono ic i uses in oden s ep esen ing he common
species sold in we ma ke s and p esen in pe idomes ic and
a m se ings. In pa allel, we de e mined se op e alence o
he same se o i uses in a coho o indi iduals wi h high
le els o occupa ional and esiden ial exposu e o oden s and
domes ic li es ock. The i uses o in e es (namely la i-
i uses, han a i uses, pox i uses, a ena i uses, and oden
pa echo i uses) we e selec ed on he basis o hei known
o p obable endemici y, hei documen ed associa ions wi h
oden -bo ne disease, and conside a ions o easibili y and
esou ces (i.e., access o e e ence s ains and well-es ablished
diagnos ic p o ocols). In addi ion o se ological sc eening,
we es ed oden li e and lung issues by RT-PCR using
gene ic p ime s o la i i uses and han a i uses).
Me hods
Roden s
Roden su eys we e conduc ed as desc ibed by Hoang
e al. (2014). B ie ly, a s we e pu chased om ma ke s in
i e p o inces o he Mekong Del a (Dong Thap, Tien Giang,
An Giang, Vinh Long, and Can Tho) du ing Oc obe o 2012,
and ield apping was conduc ed in pe idomes ic and o es
habi a s in Dong Thap p o ince du ing Ma ch o 2013.
Specimens used in his in es iga ion comp ised se a o whole
blood and issues om 275 indi idual a s (150 pu chased
in we ma ke s, 125 ield apped). Fi e oden species we e
ep esen ed: Ra us a gen i en e (n=104) Bandico a indica
(n=65), R. anezumi (n=61), R. no egicus (n=29), and
R. exulans (n=16) (Table 1).
Human se a and da a collec ion
Se a we e ob ained om a coho o 245 people li ing in
a ming communi ies in Dong Thap P o ince. E hical ap-
p o al was ob ained by he Ox o d T opical Resea ch E hics
Commi ee and he People’s Commi ee o Dong Thap P o-
ince, and he s udy was implemen ed by he p o incial
P e en i e Medicine Cen e (PMC). Pa icipan s we e eligi-
ble i hey ga e in o med consen and we e in ol ed in
aising, slaugh e ing, o p ocessing li es ock o wildli e, wi h
a minimum o 10 h exposu e pe week. Family membe s wi h
esiden ial exposu es o li es ock we e also ec ui ed (mini-
mum 1 yea o age). Phlebo omy was pe o med using s e ile
echnique by a s udy nu se. A e collec ion, all specimens
we e aliquo ed and p ese ed a -80C. In o ma ion was
collec ed on sociodemog aphic ac o s, occupa ion, house
s uc u e, hygiene, wa e sou ce, ood consump ion, house-
hold animals, de ailed animal con ac his o ies, accina ion,
and admission o hospi al in he 12 mon hs p io o he su ey.
Se ological sc eening
Human and oden se a, and oden d ied blood spo s on
il e pape s we e sc eened using an indi ec immuno luo-
escence an ibody es (IFAT) o an ibodies agains Seoul
(SEOV) and Dob a a (DOBV) han a i uses ( ep esen a i es
o a and mouse ype hos lineages, espec i ely); la i-
i uses ( ick-bo ne encephali is [TBEV] se ocomplex); a e-
na i uses (lymphocy ic cho iomeningi is i us [LCMV]);
pox i uses (cowpox i us); and pa echo i uses (Ljungan
i us). IFAT spo slides and he assay p ocedu es we e con-
duc ed as p e iously desc ibed (Kallio-Kokko e al. 2006,
Ja
¨a
¨skela
¨inen e al. 2013) using Ve o E6 cells o SEOV,
DOBV, TBEV, LCMV, and Ve o cells o cowpox and
Ljungan i uses. Vi al e e ence s ains we e 80-39 o
SEOV (An ic e al. 1991), Ano-Po oia o DOBV (Nemi o
e al. 1999), A ms ong o LCMV (Kallio-Kokko e al.
2006), Kumlinge A52 o TBEV (B umme -Ko enkon io
e al. 1973; Whi by e al. 1993), CPXV/FIN/T2000 o
cowpox i us (Pelkonen e al. 2003), and LV 145 SLG o
Ljungan (Tol e al. 2009). Se a we e dilu ed 1:20 in phos-
pha e-bu e ed saline (PBS) o ini ial sc eening, and epea
es ing wi h h ee- old se ial dilu ions was pe o med on all
human pu a i e se oposi i es. Whole blood collec ed in il e
pape s was elu ed in PBS (*1cm
2
o il e pape in 1 mL o
PBS). Fo oden se a, he seconda y an ibody was polyclonal
abbi an i-mouse luo escein iso hiocyana e (FITC) conju-
ga e dilu ed 1:30 (Dako A/S, Copenhagen, Denma k); o
human se a, he seconda y an ibody was an i-human immu-
noglobulin G (IgG) FITC conjuga e dilu ed 1:100 (Jackson
Immuno Resea ch Labo a o ies, Wes G o e, PA). Each slide
was examined independen ly by wo esea che s using an
Olympus BX51 mic oscope. Human se a wi h e idence o
TBEV eac i i y we e u he examined using di e en ial
IFAT sc eening in pa allel o dengue (DENV) and Japanese
encephali is i us ( JEV), as p e iously desc ibed (Ton e i
e al. 2011).
Molecula sc eening
Vi ological sc eening o la i i uses and han a i uses
was pe o med on all oden li e and lung samples. B ie ly,
1-cm
2
issue samples we e homogenized using a bead mill
Table 1. Roden Species Pu chased and T apped in he Mekong Del a, No embe , 2012, o Ma ch, 2013
Ra us
a gen i en e
Bandico a
indica
Ra us
anezumi
Ra us
no egicus
Ra us
exulans To al
All 104 65 61 29 16 275
Male/ emale 52/52 19/46 43/18 15/14 9/7 138/137
No. pu chased a s 88 44 9 9 0 150
To al oden s apped 16 21 52 20 16 125
No. apped in a ms 14 10 19 18 15 76
No. apped in o es / ields 2 11 33 2 1 49
66 CUONG ET AL.
(TissueLyse II, QIAGEN), and nucleic acid ex ac ed using
MagNA Pu e 96 Vi al NA Small Volume Ki (Roche) on an
au oma ed ex ac o (Roche). Following ex ac ion, nucleic
acid samples we e pooled by species ( i e samples pe pool).
cDNA om lung samples was sc eened o han a i us wi h
con en ional PCR (L segmen ) (Klempa e al. 2006). cDNA
om li e issues was sc eened o la i i uses using con-
sensus NS5 p ime s (Kuno e al. 1998). When pools es ed
posi i e, o iginal ex ac ions om indi idual samples we e
u he analyzed. Amplicons o he expec ed band size we e
sequenced comme cially (Mac ogene, Seoul, Ko ea).
S a is ical analyses
Compa ison o human se oposi i i y o han a i us (DOBV
and SEOV combined), pox, LCMV, and Ljungan i uses was
ca ied ou using a chi-squa ed es . Risk ac o analyses
o explo e associa ions be ween demog aphic/occupa ional
a iables, animal con ac his o ies, and se op e alence we e
in es iga ed using logis ic eg ession and mul i a iable
models (Dohoo e al. 2003). All s a is ical analyses we e
ca ied ou using R s a is ical so wa e (www. -p ojec .o g).
Resul s
Roden se ology
Among he 275 es ed oden s, se oposi i i y was de ec ed
in ou o he i e i us g oups, wi h mode a e le els o e-
ac i i y o TBEV (5.4% se oposi i e) and han a i uses (6.9%
se oposi i e), bu li le o no e idence o immunological
exposu e o cowpox (1.1%), a ena i us (0.4%), o Ljungan
i us (0%) (Table 2). Among he a s wi h TBEV an ibodies,
B. indica had he highes p e alence (12/65, 18.4% posi i e),
ollowed by R. a gen i en e (2/104, 1.9%) and R. anezumi
(1/61, 1.6%) ( p<0.001). To examine whe he he TBEV
se oposi i es ep esen ed ue eac i i y o TBEV se o-
complex i uses o c oss- eac i i y o he o he egionally
endemic la i uses, addi ional se ial i a ions ( i e dilu ions)
we e un in pa allel agains DENV and JEV o he 15 pu-
a i e TBEV se oposi i es and o i e se onega i es. The
di e en ial IFAT i a ion showed simila eac i i y o
TBEV, DENV, and JEV, and all samples es ed nega i e o
he h ee i uses a 1:320 dilu ion.
O e all se oposi i i y o han a i uses was 6.9% (19/275).
In spi e o he supposed c oss- eac i i y among mu ine
han a i uses, only h ee indi iduals we e posi i e o bo h
han a i al an igens (six posi i e o DOBV only, 10 posi i e
o SEOV only). This di e en ial eac i i y may indica e he
p esence o se e al han a i uses. Se oposi i e hos species
included R. no egicus,R. a gen i en e ,R. anezumi, and B.
indica. None o he R. exulans se a (n=16) showed an ibody
eac i i y o he es i uses.
Roden molecula sc eening
None o he 275 oden li e samples (including hose om
he 15 TBEV-se oposi i e oden s) es ed posi i e when
sc eened wi h he gene ic NS5 la i i us p ime s. F om
han a i us sc eening o lung issues, one R. a gen i en e
ma ke sample yielded an amplicon o app op ia e band size
in he han a i us con en ional RT-PCR assay. A 310-bp
sequence o he L segmen con i med he p esence o a
han a i us wi h a 95–98% ma ch o all SEOV sequences in
GenBank, he closes one being s ain Jaka a/Rn137/2000
om Indonesia (new GenBank acc. no. KP055755).
Human se ology
Se oposi i i y was de ec ed o all i e i us g oups, wi h
high le els o se oposi i es o TBEV (116/245, 47.3%),
mode a e se oposi i i y o Ljungan i us (18.8%, 46/245),
and lesse a es o se oposi i y o cowpox i us (4.5%, 11/
245), han a i uses (3.7%, 9/245), and LCMV (0.8%, 2/245)
(Table 3). Rega ding he nine indi iduals wi h han a i us
an ibodies, he e we e wo posi i e only o DOBV, i e
posi i e only o SEOV, and wo posi i e o bo h. Se -
oposi i i y o TBEV and pox i uses was associa ed wi h
inc eased age, whe eas se oposi i i y o Ljungan i us was
highe in younge indi iduals (Fig. 1). The e we e no sig-
ni ican associa ions be ween se oposi i i y and occupa ion.
TBEV an ibody de ec ion was highe in males (62.4%) e -
sus emales (36.8%) ( p<0.001). Di e en ial IFAT i a ions
agains DENV and JEV we e pe o med on a subse o 60
human se a, including 30 TBEV se oposi i es and 30 TBEV
se onega i es. O he 30 TBEV posi i e samples, 12 had
equi alen i e s o JEV and DENV, 17 had highe ela i e
Table 2. Roden Se op e alence o Fi e G oups o Roden -Bo ne Vi uses
No. o posi i e
No. o
oden s TBEV Han a i us
Cowpox
i us LCMV
Ljungan
i us
Roden sex
Male 138 4 (2.9) 9 (6.5) 2 (1.4) 1 (0.7) 0 (0)
Female 137 11 (8.0) 10 (7.3) 1 (0.7) 0 (0) 0 (0)
Roden Species
Ra us a gen i en e 104 2 (1.9) 5 (4.8) 0 (0) 0 (0) 0 (0)
Bandico a indica 65 12 (18.4) 10 (15.4) 1 (1.5) 1 (0.4) 0 (0)
Ra us anezumi 61 1 (1.6) 1 (1.6) 2 (3.3) 0 (0) 0 (0)
Ra us no egicus 29 0 (0) 3 (10.3) 0 (0) 0 (0) 0 (0)
Ra us exulans 16 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
To al 275 15 (5.4) 19 (6.9) 3 (1.1) 1 (0.4) 0 (0)
Values wi hin pa en heses a e pe cen ages.
RODENT-BORNE VIRUSES IN VIETNAM 67
i e s o JEV han DENV, and only one had a highe i e o
TBEV han o he JEV and DENV.
Associa ions be ween se op e alence
and animal con ac
O he 245 pa icipan s, he mos equen ly epo ed ani-
mal con ac ( >10 h pe week) in ol ed pigs (174/245, 71%),
poul y (175/245, 71%), and dogs (149/245, 61%) (Table 4).
Con ac wi h ducks, musco y ducks, and ca s was also
common (36%, 28%, and 37%, espec i ely). Twen y people
epo ed con ac wi h a s, mos commonly h ough wo k
ac i i ies, ood p epa a ion, o di ec consump ion. Th ee
coho membe s epo ed main aining ba oos s o p oduce
guano o e ilize . Risk ac o analysis in ol ed s a i ying
people as exposed o unexposed o he abo e-men ioned
animal species and examining associa ions wi h se -
op e alence o he i e i us g oups. O e all, people wi h ba
con ac had highe se oposi i i y o cowpox i us ( ela i e
isk [RR] 8.07, 95% con idence in e al [CI] 1.46, 44.67);
howe e , he sample size was ex emely small (n=3). Ra
con ac was associa ed wi h an ele a ed isk o LCMV se-
oposi i i y (RR 11.25, 95% CI 0.73, 173.16). Howe e ,
excep o he ela ionship be ween ba con ac and cowpox
se oposi i i y ( p<0.001), none o he abo e-men ioned as-
socia ions eached s a is ical signi icance.
Discussion
Roden s a e known o hos a wide spec um o human i al
and bac e ial pa hogens; howe e , in es iga ions o oden -
bo ne diseases a e ela i ely a e, and his di e se g oup o
FIG. 1. Human se op e alence o la i i us (TBEV), pa echo i us (Ljungan i us), and pox i us (cowpox i us) s a i ied
by age g oup. Coho age g oups we e 4–18 yea s (n=37), 19–38 yea s (n=86), 39–59 yea s (n=89), and 60–76 yea s
(n=33). Ba s indica e 95% con idence in e als.
Table 3. Human Se op e alence o Fi e G oups o Roden -Bo ne Vi uses
No. o posi i e
No. o
humans TBEV Han a i us
Cowpox
i us LCMV
Ljungan
i us
Coho sex
Male 101 63 (62.4) 5 (4.9) 7 (6.9) 2 (1.9) 27 (26.7)
Female 144 53 (36.8) 4 (2.7) 4 (2.7) 0 (0) 19 (13.2)
Coho occupa ion
Fa me 181 88 (48.6) 6 (3.3) 8 (4.4) 1 (0.6) 31 (17.1)
Animal heal h wo ke 29 14 (48.3) 1 (3.4) 1 (3.4) 1 (3.4) 5 (17.2)
Pig slaugh e e 12 4 (33.3) 1 (8.3) 1 (8.3) 0 (0) 4 (33.3)
Poul y slaugh e e 18 7 (38.8) 1 (5.6) 1 (5.6) 0 (0) 4 (22.2)
Ra ade 5 3 (60.0) 0 (0) 0 (0) 0 (0) 2 (40.0)
To al 245 116 (47.3) 9 (3.7) 11 (4.5) 2 (0.8) 46 (18.8)
Values wi hin pa en heses a e pe cen ages.
68 CUONG ET AL.
Table 4. Risk Fac o Analysis o Human S op e alence o Fi e Vi us G oups and Rela ed Animal Con ac Beha io s
Vi us Pig Chicken Duck Musco y Duck Dog Ca Ba Ra
TBEV
No. pos./exposed 85/174 86/175 43/88 36/69 72/149 41/91 0/3 9/20
No. pos./unexposed 31/71 30/70 73/157 80/176 44/96 75/154 116/242 107/225
RR 1.12 1.15 1.05 1.15 1.05 0.93 (NC) 0.95
95% CI (0.82, 1.52) (0.84, 1.56) (0.80, 1.38) (0.87, 1.52) (0.80, 1.39) (0.70, 1.22) (0.57, 1.57)
DOBV o SEOV
No. pos./exposed 6/174 6/175 3/88 3/69 4/149 3/91 0/3 1/20
No. pos./unexposed 3/71 3/70 6/157 6/176 5/96 6/154 9/242 8/225
RR 0.82 0.80 0.89 1.28 0.52 0.85 (NC) 1.41
95% CI (0.21, 3.17) (0.20, 3.11) (0.23, 3.48) (0.33, 4.96) (0.14, 1.87) (0.22, 8.21) (0.19, 10.69)
Cowpox i us
No. pos./exposed 8/174 8/175 4/88 5/69 8/149 4/91 1/3 1/20
No. pos./unexposed 3/71 3/70 7/157 6/176 3/96 7/154 10/242 10/225
RR 1.09 1.07 1.02 2.13 1.72 0.97 8.07 1.12
95% CI (0.30, 3.98) (0.29, 3.90) (0.31, 3.39) (0.67, 6.74) (0.47, 6.32) (0.29, 3.21) (1.46, 44.67) (0.15, 8.35)
LCMV
No. pos./exposed 1/174 1/175 1/88 0/69 1/149 1/91 0/3 1/20
No. pos./unexposed 1/71 1/70 1/157 2/176 1/96 1/154 2/242 1/225
RR 0.41 0.41 1.78 (NC) 0.64 1.69 (NC) 11.25
95% CI (0.03, 6.43) (0.03, 6.40) (0.11, 28.17) (0.04, 10.18) (0.11, 26.73) (0.73, 173.16)
Ljungan i us
No. pos./exposed 30/174 30/175 17/88 13/69 24/149 18/91 1/3 4/20
No. pos./unexposed 16/71 16/70 29/157 33/176 22/96 28/154 45/242 42/225
RR 0.77 0.75 1.05 1.0 0.70 1.09 1.79 1.07
95% CI (0.45, 1.31) (0.44, 1.29) (0.61, 1.79) (0.56, 1.79) (0.42, 1.18) (0.64, 1.85) (0.35, 9.08) (0.43, 2.68)
TBEV, ick-bo ne encephali is i us; pos., posi i e; RR, ela i e isk; CI, con idence in e al; DOBV, Dob a a i us; SEOV, Seoul i us; LCMV, lymphocy ic cho iomeningi is i us.
69

in ec ions is no equen ly p io i ized by biomedical o
public heal h esea ch communi ies. Han a i uses ( amily
Bunya i idae) a e a guably he oden -bo ne i uses o
g ea es public heal h sign icance in Asia, whe e hey a e
associa ed wi h an es ima ed 150,000–200,000 cases o
hemo hagic e e wi h enal synd ome (HFRS) each yea
(Bi e al. 2008). The han a i us genus comp ises a leas 24
ecognized i us species, se e al o which a e pa hogenic o
humans. Al hough han a i uses a e known o ci cula e in
Vie nam, ew clinical cases ha e been epo ed (Huong e al.
2010), and he ela i e scope o he associa ed disease bu den
emains poo ly cha ac e ized.
Ou s udy indica ed an o e all 8.3% han a i us se o-
p e alence among he i e oden species es ed om
sou he n Vie nam. This p e alence is consis en wi h p e i-
ous epo s om Vie nam (app oxima ely 2.8% and 8.6%
se oposi i i y in oden s om he Ho Chi Minh Ci y and
no he n p o inces, espec i ely) (T uong e al. 2009, Luan
e al. 2012). We ound han a i us an ibodies in all a species
wi h he excep ion o R. exulans. Despi e he ela i ely high
se op e alence o han a i uses, and he cu en unde -
s anding ha mos ese oi hos s de elop ch onic pe sis en
in ec ions (Eas e b ook e al. 2007, Vahe i e al. 2013), only
one a sample (R. a gen i en e ) was con i med o be han-
a i us posi i e, wi h con en ional PCR indica ing SEOV. R.
a gen i en e has no p e iously been iden i ied as a hos o
han a i uses, and he wo ldwide dis ibu ion o SEOV has
been almos exclusi ely associa ed wi h R. no egicus (Lin
e al. 2012).
Mu ine han a i uses a e belie ed o c oss- eac s ongly;
howe e , o ensu e de ec ion o a b oad spec um o po en ial
han a i us an ibodies, we used wo ypes o an igens (SEOV
and DOBV), ep esen ing a o mouse hos s, espec i ely. (I
should be no ed, howe e , ha hos s o DOBV, Apodemus
mice, a e no ound in Vie nam.) In e es ingly, mos o he
oden se a e ealed a iable eac i i y o DOBV and SEOV
i uses, wi h only i e o 19 (26.3%) se a es ing posi i e o
bo h, sugges ing ha mul iple han a i uses may be ci cula -
ing in he a ea. I is known om o he s udies in sou heas
Asia ha B. indica ca ies Thailand han a i us (Hugo e al.
2006), R. anezumi ca ies Se ang i us (Plyusnina e al.
2009), and R. no egicus ca ies SEOV.
Rega ding he human coho , we de ec ed se oposi i i y o
bo h SEOV (7/245, 2.9%) and DOBV (4/245, 1.6%), again
sugges ing ha a iable han a i uses may ci cula e. The e-
sul s a e consis en wi h p e ious low se op e alence a es
de ec ed om humans in no he n Vie nam (T uong e al.
2009). Risk ac o analysis did no sugges any signi ican
associa ions be ween han a i us se op e elance and con ac
pa e ns wi h any pa icula animal species. Ou esul s in-
dica e ha one o mo e han a i us species is endemic in
common a species o Vie nam; howe e , despi e ela i ely
in ense and common human exposu es o a s, human se o-
con e sions o han a i us a e no common.
The Fla i i us genus ( amily Fla i i idae) comp ises ap-
p oxima ely 70 i us species, including a numbe o impo -
an human pa hogens and a clade o species p esumed o be
ansmi ed p ima ily by oden s, o he small mammals, and
ba s ( he so-called ‘‘no known ec o ’’ g oup) (Kuno e al.
1998). Wi hin sou heas Asia, he only wo ecognized human
clinical diseases caused by la i i uses a e dengue and Jap-
anese encephali is, and in ec ions wi h ick-bo ne encepha-
li is (TBEV) ha e no been epo ed. Al hough Ixodes spp.
icks ha e been desc ibed in Vie nam (Kolonin e al. 1995),
di ec obse a ion o ec opa asi es on a s sugges s li le
p e alence o icks on a s o he Mekong Del a egion.
Ou IFAT sc eening esul s indica ed o e all TBEV se-
oposi i i y in humans o 47% and lesse a es in he oden s
o 5.4%. Gi en ha la i i uses induce li e-long immuni y,
ha dengue and JEV a e bo h highly endemic in sou he n
Vie nam (e.g., app oxima ely 53% dengue se ocon e sion in
humans by age 7, inc easing o 88% by age 13) (Thai e al.
2005), and ha JEV accina ion is now widely adminis e ed
h oughou he coun y (Yen e al. 2010), ou obse ed TBEV
immunological eac i i y in he humans was no a all su -
p ising and mos likely e lec s he no o ious c oss- eac i i y
o la i i uses (Roeh ig, 1986, Basile e al. 2013). In con as ,
he oden se o eac i i y o TBEV (15/275, 5.4%) was mo e
in iguing. Roden TBEV posi i es a e much less likely o be
due o dengue, because he p incipal dengue mosqui o ec o ,
Aedes aegyp i, is almos exclusi ely an h opophilic (Ha -
ing on e al. 2001). Roden s ha e ne e been sugges ed o
play a ole in JEV ansmission; howe e , gi en he high
le els o endemici y in a ms and pe iu ban a eas o he
Mekong (Lindahl e al. 2013), he obse ed se o eac i i y in
oden s could be explained by spillo e in ec ions om a ian
ese oi s ec o ed by a ious Culex species. Ou ailu e o
iden i y i al genomes in he li e issues o any o he 275
oden s sugges s ha he obse ed se ocon e sions we e
caused by ansien in ec ions, and/o he opism o he i us
is nonhepa o opic. The p esen esul s lea e open he pos-
sibili y ha non-DENV, non-JEV la i i uses ci cula e en-
demically wi hin oden s o he Mekong, pe haps in ol ing
ick ec o s, as has been ound in Malaysia (Smi h 1956) and
Thailand (Banc o e al. 1976).
None o emaining i us g oups included in ou su -
ey, namely cowpox i us, a ena i uses, and oden pa -
echo i uses, showed e idence o high se o eac i i y in he
oden s. Cowpox i uses belong o he genus O hopox i us
ha is immunologically c oss- eac i e and c oss-p o ec i e,
and cowpox in ec ion in oden s has been epo ed ac oss
Eu ope om a wide ange o oden species, al hough R.
no egicus and bank oles a e hough o be he p incipal
ese oi s (Essbaue e al. 2010). In he p esen s udy,
pox i us an ibody de ec ion in humans was 4.5%, and was
no ably highe among people o e 60, which may be a ib-
u able o p io smallpox accina ion. In con as , pox i us
se oposi i i y was only 1.1% in oden s; he posi i e oden s
we e B. indica and R. anezumi. No ably, we ound a sig-
ni ican associa ion be ween p esence o pox an ibody among
coho membe s and exposu e o ba s (RR 8.07, 95% CI 1.46,
44.67; p<0.05). Se op e alence o LCMV was much lowe
(human, 0.8%; oden , 0.4%) han has been obse ed om
Thailand, whe e o e all LCMV se op e alence among o-
den s in one s udy was 13.3%, mos ly in Bandico a sa ilei
(35.7%) and R. no egicus (31.5%) (Ni a pa ana e al. 2000).
Ljungan i us is a oden -bo ne pa echo i us and sus-
pec ed human pa hogen (Kli z and Niklasson 2013), and al-
hough ela i ely high se op e alence in humans has been
documen ed om no he n Eu ope (Ja
¨a
¨skela
¨inen e al. 2013),
o da e he e ha e been no s udies o Ljungan i us in Asia.
None o he Vie nam a s we e se o eac i e o Ljungan i us;
he lack o Ljungan i us se oposi i es among he Vie -
namese a s may no be su p ising, because Ljungan i us in
70 CUONG ET AL.
Eu ope has p e iously been associa ed p ima ily wi h bank o
ield oles (membe s o he A icolinae) a he han Ra us
spp. Howe e , as many as 18.8% (46/245) o human coho
membe s es ed posi i e o Ljungan i us an ibodies. The
human Ljungan se o eac i i y did no appea o be linked
o any pa icula animal exposu es. The age dis ibu ion o
se oposi i es declined wi h age, as has been obse ed om
su eys o human pa echo i uses (HPeV) in Eu ope (Ben-
schop e al. 2008). This pa e n o declining immuni y wi h
age may indica e human- o-human ansmission ia ecal–
o al ansmission, as is ypical o he known HPeVs , a he
han exposu es o a zoono ic ese oi . I should be no ed,
howe e , ha cu en knowledge o Ljungan immunology
sugges s ha he i us does no c oss- eac wi h HPeV 1, 2, o
4–6 (Ja
¨a
¨skela
¨inen e al. 2013). Ou esul s p o ide in igu-
ing sugges ions o equen human exposu es o an as-ye -
uniden i ied pa echo i us.
In summa y, ou s udy is he i s o documen he common
oden species aded in we ma ke s and sold o human
consump ion in Vie nam (p ima ily R. a gen i en e and B.
indica). Ou esul s con i m low- o-mode a e le els o en-
demic ci cula ion o han a i uses in he egion and sugges
possible ansmission o as ye uniden i ied oden la i i us
and human ansmission o a Ljungan-like pa echo i us.
Fu he explo a ions o pa hogen di e si y in oden ese -
oi s using la ge sample sizes and mo e sensi i e molecula
sc eening echniques (e.g., al e na i e degene a e p ime s o
genus-speci ic s udies, o me agenomic and deep sequencing
app oaches) will no doub unco e subs an ial addi ional
i al di e si y. Gi en he scale and scope o oden ading in
he Mekong, and indica ions ha oden consump ion may
also be inc easing in no he n Vie nam, es ablishing he ep-
idemiological ele ance o human exposu es o a i uses is
an impo an public heal h objec i e.
Acknowledgmen s
The au ho s would like o hank s a om he Sub-
Depa men o Animal heal h o Dong Thap o p o iding
logis ic assis ance in ca ying ou he s udy. Wo k was pa ly
unded by he Vie nam Ini ia i e on Zoono ic In ec ions
(VIZIONS), pa o he Wellcome T us Majo O e seas
P og amme (Uni ed Kingdom), and he Li-Ka-Shing Foun-
da ion, Sig id Juse
´lius Founda ion, and Academy o Finland.
Au ho Disclosu e S a emen
No compe ing inancial in e es s exis .
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Add ess co espondence o:
Julie E. B yan
Depa men o Vi ology
Ox o d Uni e si y Clinical Resea ch Uni
78 Giai Phong
Dong Da, Hanoi
Vie Nam
E-mail: [email p o ec ed]
72 CUONG ET AL.