Ga he e al. Ve Res (2016) 47:97
DOI 10.1186/s13567-016-0381-6
SHORT REPORT
Acu e andch onic in ec ions
wi hnonp ima e hepaci i us inyoung ho ses
The esa Ga he 1, S ephanie Wal e 2, S ephanie P aende 2, Daniel Tod 2, Ka s en Feige1, Eike S einmann2*
and Jessika M. V. Ca alle i1*
Abs ac
The ecen ly disco e ed nonp ima e hepaci i us (NPHV) na u ally in ec s ho ses and is he closes known homolog o
hepa i is C i us o da e. Wi hin a ollow-up s udy acu e ield in ec ions we e moni o ed in ou young Tho oughb ed
ho ses un il he ages o 12–13 mon hs. Se um samples we e analyzed o he p esence o NPHV RNA and an i-NPHV
NS3 an ibodies and li e speci ic pa ame e s we e e alua ed. The ou young ho ses we e no able o clea in ec-
ion, bu emained ch onically in ec ed o he en i e moni o ed ime pe iod despi e he p esence o NPHV speci ic
an ibodies.
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In oduc ion
Nonp ima e hepaci i us (NPHV) was desc ibed o in ec
ho ses in se e al coun ies wo ldwide and ep esen s a
new membe wi hin he amily Fla i i idae, genus Hepa-
ci i us [1, 2]. Among all hepaci i uses, NPHV is he clos-
es known homolog o hepa i is C i us (HCV) o da e.
HCV is a majo human pa hogen wi h app oxima ely
146 million people in ec ed wo ldwide [3]. In 75–85% o
acu ely in ec ed pa ien s p og ession o ch onic disease
occu s, which can lead o hepa ic ib osis, ci hosis and
ca cinoma [4]. In ecen yea s, simila as well as dis inc
ea u es be ween HCV and NPHV ha e been desc ibed.
NPHV is highly p e alen in ho ses wi h 30–40% se o-
posi i i y and 3% i emia [1, 5–10]. Howe e , disease
associa ion emains unce ain al hough se e al s udies
epo ed subclinical hepa i is in in ec ed ho ses. A mild
ele a ion o se um li e enzymes was obse ed a se o-
con e sion in some a ec ed ho ses, al hough in mos
ho ses se um li e enzymes emained wi hin he e e -
ence ange [7, 11, 12]. Single epo s exis abou NPHV
in ec ed ho ses su e ing om se e e hepa i is. Ye , a
causa i e ela ionship s ill needs o be con i med [12, 13].
Compa able o HCV in ec ion, hepa o opism has been
con i med o NPHV [11, 14]. Ne e heless, he ou es
o NPHV ansmission emain mos ly unclea . Simila o
HCV a pa en e al in ec ion ou e ia di ec blood–blood
con ac has been desc ibed in ho ses by expe imen al
inocula ion wi h in ec ious plasma con aining NPHV
[12]. In a ecen s udy, we in es iga ed wen y ma e- oal
pai s a pa u i ion un il 6mon hs pos pa um and could
show he occu ence o e ical ansmission o NPHV a
pa u i ion [15]. Mo eo e , we obse ed ansmission o
NPHV isola es wi hin he espec i e pas u e he ds indi-
ca ing he possibili y o a di ec ansmission be ween
ho ses. In e es ingly, we could show ha se e al oals
became i emic wi hin he i s 6 mon hs o li e [15].
To shed some ligh on he cou se o na u ally acqui ed
NPHV in ec ion o he young ho ses, we aimed o pe -
o m a ollow-up s udy by moni o ing ou oals ( oal # 9,
# 10, # 17 and # 19) un il he age o 12–13mon hs.
Ma e ials, me hods and esul s
Blood samples we e aken om hese ou oals a indi-
ca ed ime poin s (Figu e1; Table1). All samples we e
ob ained wi h ull owne consen as pa o ou ine
heal h managemen . Addi ionally, a gene al examina-
ion o he oals was conduc ed a all in es iga ed ime
poin s. Nex , se a was analyzed o he p esence o NPHV
RNA and an i-NPHV NS3 an ibodies by a SYBR G een
Open Access
*Co espondence: Eike.S einmann@ winco e.de; Jessika.Ca alle i@
iho-hanno e .de
1 Clinic o Ho ses, Uni e si y o Ve e ina y Medicine Hanno e ,
Founda ion, Bün eweg 9, 30559 Hanno e , Ge many
2 Ins i u e o Expe imen al Vi ology, TWINCORE Cen e o Expe imen al
and Clinical In ec ion Resea ch, Feodo -Lynen-S . 7, 30625 Hanno e ,
Ge many
Page 2 o 5
Ga he e al. Ve Res (2016) 47:97
AB
C
an i-NPHV NS3 an ibodies (ab)
NPHV RNA glu ama e dehyd ogenase (GLDH)
γ-glu amyl ans e ase (GGT)
aspa a e amino ans e ase (AST)
oal # 9
02468101214
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
103
104
105
106
cu -o
age o he oal in mon hs
NPHV RNA copies/ mL
an i-NPHV NS3 ab [RLU]
oal # 10
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
NPHV RNA copies/ mL
10
3
10
4
105
106
cu -o
an i-NPHV NS3 ab [RLU]
02468101214
age o he oal in mon hs
li e pa ame e s
02468101214
0
5
10
15
20
25
30
35
40
60
100
140
180
220
260
age o he oal in mon hs
GGT, GLDH (U/L)
li e pa ame e s
AST (U/L)
0
5
10
15
20
25
30
35
40
GGT, GLDH (U/L)
02 468101214
age o he oal in mon hs
60
100
140
180
220
260
AST (U/L)
oal # 17
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
NPHV RNA copies/ mL
10
3
10
4
10
5
10
6
cu -o
an i-NPHV NS3 ab [RLU]
02468101214
age o he oal in mon hs
oal # 19
D
10
3
10
4
10
5
10
6
cu -o
an i-NPHV NS3 ab [RLU]
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
NPHV RNA copies/ mL
02468101214
age o he oal in mon hs
li e pa ame e s
60
100
140
180
220
260
AST (U/L)
0
5
10
15
20
25
30
35
40
GGT, GLDH (U/L)
02 468101214
age o he oal in mon hs
li e pa ame e s
60
100
140
180
220
260
AST (U/L)
02 468101214
age o he oal in mon hs
0
5
10
15
20
25
30
35
40
GGT, GLDH (U/L)
Page 3 o 5
Ga he e al. Ve Res (2016) 47:97
based quan i a i e eal- ime PCR (qRT-PCR) and luci -
e ase immunop ecipi a ion assay (LIPS), espec i ely, as
desc ibed ea lie [1, 11, 15]. As shown in Figu e1, NPHV
RNA was de ec ed in he se um o all oals 6mon hs a e
bi h. F om he ini ial i us de ec ion onwa ds he ou
acu ely in ec ed young ho ses emained i emic wi h high
i al loads du ing he en i e moni o ed 7mon hs and none
o hem elimina ed he i us un il he las sampling a he
age o 12–13mon hs (Figu e1A–D). Fo he oals # 9, #
10 and # 19 an i-NPHV NS3 an ibodies we e de ec ed a
he ime poin o bi h, dec eased un il 6mon hs pos pa -
um below o close o he cu -o o he assay and NPHV
speci ic an ibodies appea ed again a e NPHV RNA was
de ec ed in he se um (Figu e1A, B and D). O no e, oal
# 17 was he only oal no ecei ing ma e nal an i-NPHV
NS3 an ibodies a e oaling and became newly in ec ed
a a simila age as he emaining oals and p oduc ion o
an i-NPHV NS3 an ibodies was obse ed om he age o
10mon hs onwa ds (Figu e1C). In Table1 de ails on he
NPHV RNA and an i-NPHV NS3 an ibody s a us o he
ollow-up samples o he ou ho ses a e gi en (Table1).
To in es iga e he clinical ele ance o NPHV in ec ion
o li e disease, he ho ses we e clinically examined and
blood was analyzed o he li e speci ic enzymes glu a-
ma e dehyd ogenase (GLDH), γ-glu amyl- ans e ase
(GGT) and aspa a e amino ans e ase (AST) in he labo-
a o y o he small animal clinic a he Uni e si y o Ve -
e ina y Medicine Hanno e , Founda ion. Resul s o each
sampling ime poin a e shown in Figu e1, lowe panels.
A mild ele a ion o li e enzymes was obse ed a he age
o 12–13mon hs o oals # 9, # 17, and # 19. Howe e ,
alues o all o he ime poin s emained wi hin he e e -
ence ange and no clinical signs indica ing li e disease
we e ound a any sampling ime poin .
In conclusion, ou acu ely in ec ed oals de eloped
ch onic in ec ion and emained i emic wi h high i al
loads du ing he en i e moni o ed 7mon hs. Despi e he
p esence o an i-NPHV NS3 an ibodies, he ou young
ho ses we e no able o clea he in ec ion wi hin he
moni o ed ime pe iod.
Discussion
This s udy p o ides he i s desc ip ion o he clinical
cou se o na u ally acqui ed NPHV in ec ion in young
ho ses. This ollow-up s udy was conduc ed in he con ex
o a p e ious s udy in es iga ing he occu ence o e ical
ansmission o NPHV in a ho se coho , whe e we moni-
o ed wen y Tho oughb ed b oodma es and hei oals
om pa u i ion un il 6mon hs a e oaling [15]. He e,
we moni o ed ou o hese oals o ano he 7mon hs.
These oals became NPHV RNA posi i e a he age o
6mon hs and s ayed i emic o ano he 7mon hs wi h
he concu en de ec ion o an i-NPHV NS3 an ibodies.
The ac i i y o li e speci ic enzymes inc eased in h ee
oals a he ages o 12–13mon hs. The mild ele a ion o
enzyme ac i i y could indica e a subclinical hepa i is due
o he pe sis en NPHV in ec ion. Fo eliable diagnosis
li e biopsies would be equi ed, since he ele a ion o
li e enzymes migh ha e had an un ela ed eason.
(See igu e on p e ious page.)
Figu e1 Cou se o in ec ion in he ou oals # 9, # 10, # 17 and # 19 om pa u i ion un il he ages o app oxima ely 13 mon hs.
Se um samples om he i s h ee sampling ime poin s (a pa u i ion and a he ages o 3 and 6 mon hs a e de i ed om a p e ious s udy
(ma ked wi h a g ey backg ound) [15]. Un il he ages o 12–13 mon hs hese oals we e u he moni o ed wi hin his ollow-up s udy and se um
was aken a h ee addi ional ime poin s. All se um samples we e analyzed o he p esence o an i-NPHV NS3 an ibodies (ab) (g ey bulle ) and
NPHV RNA (black squa e) by LIPS and qRT-PCR, espec i ely. The cu -o limi o he LIPS was de e mined by he mean alue o wells con aining only
bu e A, he RUC-NS3 usion p o ein and A/G beads plus h ee s anda d de ia ions and is illus a ed as dashed line. In he lowe panels, li e speci ic
pa ame e s (GLDH, GGT and AST) a e shown o each oal a he h ee ollow-up ime poin s as un illed symbols, wi h he ollowing e e ence anges
se by he labo a o y: GLDH < 6 U/L, GGT < 20 U/L and AST < 170 U/L. (A) Foal # 9, (B) oal # 10, (C) oal # 17 and (D) oal # 19.
Table 1 Raw alues o NPHV RNA and an i-NPHV NS3 an i-
bodies o he acu ely in ec ed oals
All collec ed ollow-up se um samples we e analyzed o he p esence o NPHV
RNA and an i-NPHV NS3 an ibodies (ab) by qRT-PCR and LIPS, espec i ely. NPHV
RNA i e s a e displayed as RNA copies/mL. An i-NPHV NS3 ab alues a e gi en
as RLU, whe eas alues abo e he cu -o a e highligh ed in i alic numbe s.
Follow-ups samples
Sample
# I Sample
# II Sample
# III
Foal # 9
Age in mon hs 8 10 13.5
An i-NPHV NS3 ab (RLU) 6.57E + 04 2.06E + 05 2.18E + 05
NPHV RNA (copies/mL) 8.67E + 06 4.45E + 06 9.27E + 06
Foal # 10
Age in mon hs 7.5 9.5 13
An i-NPHV NS3 ab (RLU) 8.19E + 03 3.50E + 04 4.86E + 04
NPHV RNA (copies/mL) 2.15E + 07 6.88E + 06 5.81E + 07
Foal # 17
Age in mon hs 7 9.5 13
An i- PHV NS3 ab (RLU) 4.07E + 03 1.17E + 04 2.43E + 04
NPHV RNA (copies/mL) 5.44E + 07 1.20E + 07 4.36E + 07
Foal # 19
Age in mon hs 6.5 8.5 12
An i-NPHV NS3 ab (RLU) 2.12E + 04 6.45E + 04 8.68E + 04
NPHV RNA (copies/mL) 3.25E + 07 3.17E + 06 6.44E + 07
Page 4 o 5
Ga he e al. Ve Res (2016) 47:97
In a e ospec i e s udy Ma suu e  al. also de ec ed
NPHV in ec ions in 5 ou o 7 young ho ses aged
be ween 4 and 6mon hs and 2yea s [16], suppo ing he
ime o in ec ion in ou oals. In h ee oals ( oal # 9, #
10, and # 19) ma e nal an i-NPHV NS3 an ibodies we e
de ec ed a e bi h un il h ee o 6mon hs pos pa um
(Figu es1A, B and D). One could specula e ha a e
deg ada ion o ma e nal an ibodies, he oals a e mo e
suscep ible o a NPHV in ec ion. Howe e , oal # 17
did no ecei e p o ec ion by ma e nal an ibodies, ye i
became in ec ed a he same age as he h ee o he oals.
Mo eo e , i is no clea whe he he ma e nal an ibod-
ies p o ec agains a NPHV in ec ion. So a , he cou se
o in ec ion o NPHV in young ho ses has only been
desc ibed by Ramsay e al. who expe imen ally in ec ed
wo oals a he age o 2–4weeks and moni o ed hem
o mo e han 1 yea pos -in ec ion [12]. These oals
became i emic sho ly a e inocula ion and emained
pe sis en ly in ec ed o he en i e moni o ed 63weeks.
An i-NPHV an ibodies we e de ec ed om he age o
app oxima ely 8 weeks onwa ds and peaked 23 weeks
pos -in ec ion. In he ollowing weeks, he de ec ion o
an ibodies dec eased in pa allel wi h he i al loads ha
sha ply declined om 40weeks pos -in ec ion onwa ds.
In e es ingly, in ou s udy ou na u ally in ec ed oals
also emained pe sis en ly in ec ed du ing he en i e
moni o ed ime wi h high i al loads and we e no able
o clea he in ec ion despi e he p esence o an ibodies
om he age o 7 o 9mon hs onwa ds. The eason o
he inabili y o he ou young ho ses o elimina e NPHV
emains unclea . The de elopmen o he immune sys-
em o young ho ses is only pa ially unde s ood. I is
known, ha he onse o he adap i e immune esponse
is delayed and an ibody esponses o ype IgG4, IgG7 and
IgE as well as T cell and cy okine esponses slowly e ol e
wi hin he i s yea o li e [17]. Howe e , o unde -
s and he complex in e ac ion o NPHV and he equine
immune sys em, u he esea ch is equi ed. I has been
shown, ha adul ho ses a e o en able o clea NPHV
in ec ion wi hin 2mon hs a e acu e in ec ion al hough
pe sis en ly in ec ed ho ses ha e also been desc ibed
analogous o ch onic HCV in ec ion in humans [11, 12].
In conclusion, in ou young ho ses he NPHV ield
in ec ion p og essed om acu e o ch onic in ec ion. The
ou ho ses we e no able o clea NPHV un il he ages
o 12–13mon hs despi e he p esence o an ibodies o
se e al mon hs.
Abb e ia ions
AST: aspa a e amino ans e ase; GGT: γ-glu amyl ans e ase; GLDH: glu a-
ma e dehyd ogenase; HCV: hepa i is C i us; LIPS: luci e ase immunop ecipi a-
ion sys em; NPHV: nonp ima e hepaci i us; NS 3: nons uc u al p o ein 3; RLU:
ela i e ligh uni s; qRT-PCR: quan i a i e eal- ime polyme ase chain eac ion.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Au ho s’ con ibu ions
All au ho s pa icipa ed in he design o he s udy. TG conduc ed he expe i-
men s, analyzed he da a and p epa ed he manusc ip . SW, SP, DT, KF, ES and
JMVC ha e e ised he manusc ip and coo dina ed he esea ch. All au ho s
ead and app o ed he inal manusc ip .
Acknowledgemen s
We a e g a e ul o S i ung Ges ü Fäh ho , especially o S e an Ull ich and
Da id Sachs, o hei g ea suppo o ou s udy. We also hank Pe e D.
Bu belo (NIH, Ma yland, USA) o p o iding he Renilla-luci e ase-NS3 usion
plasmid and all membe s o he Ins i u e o Expe imen al Vi ology, Twin-
co e, o help ul sugges ions and discussions. We a e also hank ul o Anja
Seemann-Jensen and Ch is iane Rich e o echnical suppo .
Funding
ES was suppo ed by he Helmhol z Cen e o In ec ion Resea ch and TG
ecei ed inancial suppo om a s ipend o he Clinic o Ho ses, Uni e si y o
Ve e ina y Medicine Hanno e .
Recei ed: 26 July 2016 Accep ed: 2 Sep embe 2016
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