Non-primate hepacivirus (NPHV) is a recently identified hepacivirus (family Flaviviridae) in dog and horse; however, the disease associations remain unknown. This study reports the detection of natural NPHV infection in a horse with apparent hepatitis, liver damage and high-level viraemia. NPHV could be hepatotropic and associated with hepatitis in horses

Boros, Ákos

Maza, Norbert

Pankovics, Péter

Reuter, Gábor

Non-primate hepacivirus (NPHV) is a recently identified hepacivirus (family Flaviviridae) in dog and horse; however, the disease associations remain unknown. This study reports the detection of natural NPHV infection in a horse with apparent hepatitis, liver damage and high-level viraemia. NPHV could be hepatotropic and associated with hepatitis in horses.</jats:p

Gábor Reuter

Norbert Maza

Péter Pankovics

Ákos Boros

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Acta Veterinaria Hungarica

Non-primate hepacivirus infection with apparent hepatitis in a horse — Short communication

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Acta Veterinaria Hungarica 62 (3), pp. 422–427 (2014) DOI: 10.1556/AVet.2014.011 First published online 19 July 2014 0236-6290/$ 20.00 © 2014 Akadémiai Kiadó, Budapest NON-PRIMATE HEPACIVIRUS INFECTION WITH APPARENT HEPATITIS IN A HORSE – SHORT COMMUNICATION Gábor REUTER1*, Norbert MAZA2, Péter PANKOVICS1 and Ákos BOROS1 1Regional Laboratory of Virology, National Reference Laboratory of Gastroenteric  Viruses, ÁNTSZ Regional Institute of State Public Health Service, Szabadság út 7,  H-7623 Pécs, Hungary; 2Directorate of Food Chain Safety and Animal Health,  Government Office for Baranya County, Pécs, Hungary (Received 24 October 2013; accepted 12 March 2014) Non-primate hepacivirus (NPHV) is a recently identified hepacivirus (fam-ily Flaviviridae) in dog and horse; however, the disease associations remain un-known. This study reports the detection of natural NPHV infection in a horse with apparent hepatitis, liver damage and high-level viraemia. NPHV could be hepato-tropic and associated with hepatitis in horses. Key words: Non-primate hepacivirus, hepacivirus, Flaviviridae, hepatitis, viraemia, horse The genus Hepacivirus, one of the four genera in the family Flaviviridae, comprises the assigned prototype species, hepatitis C virus (HCV) and an unas-signed GB virus B (GBV-B) (Simmonds et al., 2012). Although the evolutionary origin of human hepatitis C virus remains undetermined (Simmonds, 2013), a close non-primate homologue of this virus (now called non-primate hepacivirus, NPHV) has been recently identified in dogs in the USA (Kapoor et al., 2011) and in horses in the USA (Burbelo et al., 2012), UK (Lyons et al., 2012) and Ger-many (Drexler et al., 2013). Canine hepacivirus (CHV) was detected in the respi-ratory tract with a potential association with respiratory disease (Kapoor et al., 2011). NPHV RNA was detected in 8 (7.8%) of 103, 3 of 142 (2.1%) and 7 of 210 (3.3%) plasma samples collected from healthy horses in the USA, UK and Germany (Burbelo et al., 2012; Lyons et al., 2012; Drexler et al., 2013). In addi-tion, results of a novel serological test for antibodies to the conserved nonstruc-tural protein 3 (NS3) showed a seropositivity of 35% among these horses in the USA (Burbelo et al., 2012). Disease associations of NPHV in horses remain un-known. Clinical records of the three infected horses in the UK failed to identify evidence of hepatitis or systemic disease; however, a followed animal was vi-raemic during the 5-month follow-up period (Lyons et al., 2012).                                                  *Corresponding author; E-mail: reuter.gabor@ddr.antsz.hu; Phone: 0036 (72) 514-979; Fax: 0036 (72) 514-949  SUSPECTED NON-PRIMATE HEPACIVIRUS HEPATITIS IN A HORSE 423 Acta Veterinaria Hungarica 62, 2014 This study reports the detection of natural NPHV infection in a horse with apparent hepatitis in Hungary. NPHV could be associated with hepatitis in horses.  Materials and methods Blood samples were collected on 1 March, 2 April, 26 May and 13 August 2013 from an 11-year-old male domestic horse (DH1) living at a family stall in Baranya County, Hungary. Liver function tests (Table 1) were done for bio-chemical confirmation and follow-up of hepatitis and hepatic insufficiency. Blood samples were separated by centrifugation and frozen at –80 °C until re-verse transcription-PCR (RT-PCR) testing. Specific primer pairs (EQNS5BIS/ EQNS5BOIAS) were used for detection of nonstructural protein NS5B of NPHV by RT-PCR described previously (Lyons et al., 2012) (Table 1). Specific reverse or forward primers were designed based on the available NPHV sequences to de-termine the complete viral genome by RT-PCR (Table 1). Specific primer pairs were also used for excluding two novel equine pegivirus (family Flaviviridae) infections [Theiler’s disease-associated virus (TDAV) and equine pegivirus (EPgV)] in horse by RT-PCR published recently (Chandriani et al., 2013; Ka-poor et al., 2013). PCR products were sequenced directly (BigDye Reaction Kit, Applied Biosystems, Warrington, UK) using the PCR primers and run on an automated sequencer (ABI PRISM 310 Genetic Analyzer, Applied Biosystems, Stafford, Texas, USA). Table 1 Biochemical characteristics, non-primate hepacivirus (NPHV) detection and quantification in se-rum pairs collected from a domestic horse (DH1) with clinical jaundice in acute (sera 1 and 2) and convalescent (sera 3 and 4) phases Domestic horse (DH1)  Serum 1 (1 March 2013)Serum 2 (2 April 2013) Serum 3 (26 May 2013) Serum 4 (13 August 2013)Reference  range in horse (Latimer et al., 2003) Bilirubin (mg/dl) 19.1 27.7 17.1 16.7 0–3.2 GOT (IU/l) 968 231 276 291 160–412 GPT (IU/l) 21 4 8 9 2.7–21 ALP (IU/l) 1529 243 170 162 70–227 LDH (IU/l) 708 713 596 902 112–456 GGT (IU/l) 105.2 64 22 19 6–32 RT-PCR (NPHV) NA +++ + + – Real-time PCR (Ct) NA 20.97 23.72 23.71 – Elevated liver function test results are indicated with bold letters. GOT: glutamate-oxaloacetate transaminase; GPT: glutamic-pyruvic transaminase; ALP: alkaline phosphatase; LDH: lactate de-hydrogenase; GGT: gamma-glutamyl transferase; RT-PCR: reverse transcription-polymerase chain reaction; Ct: crossing threshold; NA: not available 424 REUTER et al. Acta Veterinaria Hungarica 62, 2014 Reference sequences were obtained from GenBank and aligned using Clustal X, then similarity calculations were performed by GeneDoc. Phyloge-netic tree of the amino acid alignment of nonstructural protein 3 (NS3) was cre-ated using the neighbour-joining method based on the Jones-Taylor-Thornton matrix-based model of MEGA5 (Tamura et al., 2011). RNA quantification relied on primers (NPHV-5UTR-F: 5’-CCACGGTCTGATAGGATGCTT correspond-ing to nt 329 to 349 of JQ434002; NPHV-5UTR-R: 5’-GCCCGCGGATGGCCT TCTT corresponding to nt 523 to 541 of JQ434002) designed for the highly con-served 5’-untranslated region (UTR) using real-time PCR assay (LightCycler FastStart DNA Master SYBR Green I, Roche, Mannheim, Germany). The com-plete genome sequence of NPHV found in the Hungarian horse has been submit-ted to GenBank under the accession number KF177391.   Results On 1 March 2013, during a veterinary visit, a domestic horse (DH1) show-ing severe jaundice (conjunctival icterus) of unknown origin was found in a fam-ily stall in Baranya County, Hungary. DH1 had no history of jaundice or intrave-nous administration of transfusion or passive immunisations in the previous year; however, it was vaccinated yearly with inactivated influenza/tetanus (Duvaxyn IE-T Plus, Fort Dodge AH) and West Nile virus (Duvaxyn WNV, Fort Dodge AH) vaccines. Liver function tests confirmed hepatitis and hepatic insufficiency (Table 1). Routine microscopic examination of a blood smear was negative for Babesia, microfilariae, Ehrlichia, Trypanosoma and Anaplasma. Microagglutina-tion (OIE MM 2008:2.1.9.B.2.a) and agar immunodiffusion (OIE MM 2008:2.5.6.B.2.a) tests were negative for leptospirosis (Leptospira hardjo, L. grippotyphosa, L. icterohaemorrhagiae, L. tarassovi and L. pomona) and equine infectious anaemia virus (EIAV) infections, respectively (http://www.oie.int/ international-standard-setting/terrestrial-manual/access-online). When the possi-bility of NPHV infection was revealed, another blood sample (serum 2) was col-lected and tested strongly positive for NPHV by conventional RT-PCR. For a follow-up study, blood samples were collected again on 26 May and 13 August 2013, when the intensity of the jaundice had clinically decreased. These sera showed a lower level of positivity for NPHV by RT-PCR (Table 1). NPHV viral loads were parallel measured in the sera by real-time PCR (Table 1). The cross-ing threshold (Ct) was considerably lower in the acute than in the convalescent stage, indicating a higher amount of NPHV during the clinical phase (Table 1). Complete NPHV viral genome (9219 nt + polyA) of strain NPHV-DH1/2013/HUN (KF177391) was characterised from serum 2. The Hungarian NPHV clustered phylogenetically with the previously described equine hepaciviruses. Figure 1 shows the phylogenetic analysis of NPHV-DH1/2013/HUN and hepaciviruses  SUSPECTED NON-PRIMATE HEPACIVIRUS HEPATITIS IN A HORSE 425 Acta Veterinaria Hungarica 62, 2014 based upon the amino acid sequences of NS3 (nt 3545 to 5523 in the EpgV; KC410872) (Fig. 1). Compared to the available complete genome sequences of NPHV (n = 10), the DH1 nucleotide differences (10–17%) represented mostly synonymous mutations, resulting in amino acid distances of 3–6%. Sera 2–4 of DH1 were negative for Theiler’s disease-associated virus (TDAV) and equine pegivirus (EPgV) by RT-PCR.  Fig. 1. Phylogenetic analysis (neighbour-joining method, MEGA version 5.1) of NPHV-DH1/2013/HUN (KF177391; bold letters) and other representative hepaciviruses including the available NPHV genomes in GenBank based upon the amino acid sequences of nonstructural pro-tein 3 (NS3) (from nt 3545 to nt 5523 in the EpgV genome, KC410872). HCV: hepatitis C virus; NPHV: non-primate hepacivirus; GBV-B: GB virus B; EPgV: equine pegivirus. Equine pegivirus was used as outgroup to root the tree   Discussion Although the recently identified NPHV is a non-primate homologue of hepatitis C virus causing hepatitis in humans, the disease associations of NPHV in horses remain unknown. This study reports the detection of a natural NPHV infection in a horse with apparent hepatitis. Both the clinical signs and the liver function tests provided a clear indication of liver damage. The first-round PCR was already positive for the detection of NPHV, and therefore the nested-PCR 426 REUTER et al. Acta Veterinaria Hungarica 62, 2014 assay was not necessary as described by Lyons et al. (2012), which indicates a high amount of viral nucleic acid in sera. In addition, the high level of viraemia was also confirmed by the low level of crossing threshold (Ct) by real-time PCR. The laboratory test results (viral nucleic acid level, liver function test) changed parallel to the clinical findings (intensity of jaundice) during the follow-up inves-tigation. Although sera obtained from DH1 4.5 months apart were viraemic, in-dicating persistent infection, during clinical recovery the viral nucleic acid level was lower and most of the liver function test results returned to the normal refer-ence ranges. Exclusion of the presence of two novel pegiviruses (TDAV and EPgV) in family Flaviviridae, which are likely (TDAV) and possibly (EPgV) as-sociated with liver infection and disease in horses (Chandriani et al., 2013; Ka-poor et al., 2013) strengthen the case for a link between NPHV and liver disease. Although the source and the time of NPHV infection (acute or a reactiva-tion of a chronic, clinically inapparent infection?) remains unknown, non-primate hepacivirus could be hepatotropic and associated with symptomatic hepatitis in horses.  Acknowledgements This work was supported by a grant from the Hungarian Scientific Research Fund (OTKA, K83013). G. R. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. We thank Dr. Tamás Kőszegi (University of Pécs, Pécs, Hungary) and Dr. Csaba Nemes (National Food Chain Safety Office, Kaposvár, Hungary) for their help with the liver function tests and the routine microbiological diag-nostic tests, respectively.   References Burbelo, P. D., Dubovi, E. J., Simmonds, P., Medina, J. L., Henriquez, J. A., Mishra, N., Wagner, J., Tokarz, R., Cullen, J. M., Ladarola, M. J., Rice, C. M., Lipkin, J. M. and Kapoor, A. (2012): Serology-enabled discovery of genetically diverse hepaciviruses in a new host. J. Virol. 86, 6171–6178. Chandriani, S., Skewes-Cox, P., Zhong, W., Ganem, D. E., Divers, T. J., Van Blaricum, A. J., Ten-nat, B. C. and Kistler, A. L. (2013): Identification of a previously undescribed divergent vi-rus from the Flaviviridae family in an outbreak of equine serum hepatitis. Proc. Natl Acad. Sci. USA 110, 1407–1415. Drexler, J. F., Corman, V. M., Müller, M. A., Lukashev, A. N., Gmyl, A., Coutard, B., Adam, A., Ritz, D., Leijten, L. M., van Riel, D., Kallies, R., Klose, S. M., Gloza-Rausch, F., Binger, T., Annan, A., Adu-Sarkodie, Y., Oppong, S., Bourgarel, M., Rupp, D., Hoffmann, B., Schlegel, M., Kümmerer, B. M., Krüger, D. H., Schmidt-Chanasit, J., Setién, A. A., Cot-tontail, V. M., Hemachudha, T., Wacharapluesadee, S., Osterrieder, K., Bartenschlager, R., Matthee, S., Beer, M., Kuiken, T., Reusken, C., Leroy, E. M., Ulrich, R. G. and Drosten, C. (2013): Evidence of novel hepaciviruses in rodents. PLoS Pathog. 9, e1003438. Kapoor, A., Simmonds, P., Cullen, J. M., Scheel, T. K. H., Medina, J. L., Giannitti, F., Nishiuchi, E., Brock, K. V., Burbelo, P. D., Rice, C. M. and Lipkin, W. I. (2013): Identification of a pegivirus (GB virus-like virus) that infects horses. J. Virol. 87, 7185–7190.  SUSPECTED NON-PRIMATE HEPACIVIRUS HEPATITIS IN A HORSE 427 Acta Veterinaria Hungarica 62, 2014 Kapoor, A., Simmonds, P., Gerold, G., Qaisar, N., Jain, K., Henriquez, J. A., Firth, C., Hirschberg, D. L., Rice, C. M., Shields, S. and Lipkin, W. I. (2011): Characterization of a canine ho-molog of hepatitis C virus. Proc. Natl Acad. Sci. USA 108, 11608–11613. Latimer, K. S., Mahaffey, E. A. and Prasse, K. W. (2003): Duncan and Prasse’s Veterinary Labora-tory Medicine: Clinical Pathology, 4th edition. Wiley-Blackwell, Ames, IA, USA. Lyons, S., Kapoor, A., Sharp, C., Schneider, B. S., Wolfe, N. D., Culshaw, G., Corcoran, B., McGorum, B. C. and Simmonds, P. (2012): Nonprimate hepaciviruses in domestic horses, United Kingdom. Emerg. Infect. Dis. 18, 1976–1982. Simmonds, P. (2013): The origin of hepatitis C virus. Curr. Top. Microbiol. Immunol. 369, 1–15. Simmonds, P., Becher, P., Collett, M. S., Gould, E. A., Heinz, F. X., Meyers, G., Monath, T., Plet-nev, A., Rice, C. M., Stiasny, K., Thiel, H. J., Weiner, A. and Bukh, J. (2012): He-pacivirus. In: King, A. M. Q., Adams, M. J., Carstens, E. B. and Lefkowitz, E. J. (eds) Vi-rus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the Interna-tional Committee on Taxonomy of Viruses, Elsevier, San Diego. pp. 1014–1020. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011): MEGA5: Mo-lecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739.   

Non-primate hepacivirus infection with apparent hepatitis in a horse — Short communication

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