Acute and chronic infections with nonprimate hepacivirus in young horses.

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. © 2016 The Au ho (s). This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional 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 you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/ publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. 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 Re e ences 1. 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