FRANK J. MILNE STATE-OF-THE-ART LECTURE


viruses to be reported.99 NPHV was found in pooled equine serum and in the serum of eight of 103 normal horses (7.8%), of which 35% were NPHV- antibody positive; samples tested were those sub- mitted for equine infectious anemia (EIA) testing at Cornell, and it was therefore assumed that horses were healthy. Since that description in the United States, NPHV viremia has been reported in three of 142 horses (2.1%) in Great Britain and in seven of 210 horses (3.3%) from Germany.103,104 Multiple NPHV infections have also been reported in Japan and Brazil.105,106 Similar incidence of NPHV infec- tion in clinically healthy horses is found in France (Stephane Pronost, personal communication). Cur- rently, accumulated data from Europe, North Amer- ica, and Asia suggests that nearly 40% of all adult horses are seropositive, prevalence of active infec- tion is 4% and approximately one fifth of those 4% are chronic carriers.103–105 No infections from ei- ther of these three new viruses (NPHV, TDAV, EPgV) have been found in mules or don- keys. NPHV is a member of the Hepacivirus genus in which human HCV are the prototype members and of the three new viruses found in horses, NPHV is most closely related genetically to HCV. NPHV has also been found commonly in bats, rodents, and Old World moneys and it is the only one of the three recently discovered horse hepatitis viruses known to be hepatotropic in the horse.102,107 As regards clin- ical importance, the testing of a somewhat random assortment of naturally infected horses has sug- gested that there is little documented association between NPHV infection and clinical hepatic dis- ease in those horses.103,108 In NPHV experimen- tally infected horses there is mild disease documented by elevations in liver enzymes and lym- phocytic portal inflammation and piecemeal hepato- cyte necrosis.107,109 The elevations seem to occur shortly after development of measureable antibod- ies, suggesting that antibody-mediated activity re- lated to the virus could cause some disease. When severe combined immunodeficient foals were exper- imentally infected,109 they produced no antibody and there was no increase in GGT, further support- ing an immune response to the infection and asso-


civirus (NPHV; also called hepacivirus), Theiler’s disease–associated virus (TDAV) and equine pegivi- rus (EPgV), which are all close Flaviviridae family relatives of human hepatitis C viruses (HCV).99–101 The horse seems to be unique among our domestic animal for harboring these so-called “hepatitis vi- ruses.”102 The recent discovery of these equine hepatitis viruses is directly related to the use of the newer technology involving nucleic acid deep se- quencing of samples. Unknown nucleotides are then reassembled using de novo computerized se- quencing to identify new agents. One of the prob- lems with this technology, for us as clinicians, is that finding new and infectious agents may not be clini- cally important. NPHV was the first of the three new blood borne


78 2015  Vol. 61  AAEP PROCEEDINGS


ciation with disease.109 The only report of clinical disease associated with NPHV infection is a case report from Europe of Theiler’s disease with the horse having high levels of NPHV viremia and as the viremia diminished the horse recovered.110 Although our data set is small, horses that are chronically infected with NPHV do not seem to de- velop clinical disease. We have one horse at Cor- nell that has retrospectively from frozen and stored samples been infected for 10 years (thanks to Dr. Doug Antczak for storing serum for such a long time). This horse has no elevations in liver en- zymes and a liver biopsy obtained after at least 9 years of infection was normal. Most horses infected with NPHV seem to have relatively slow develop- ment of NPHV specific antibody (approximately 6–8 weeks)107,109 followed by viral clearance in the fol- lowing months. In 2014, with the collaboration of scientists at both Rockefeller and Columbia Univer- sities, we were able to successfully infect a seroneg- ative horse with infectious clones of NPHV, yielding high RNA titers in the serum and liver of the horse and establishing the molecular components of a functional NPHV genome; cell culture of HCV like viruses is extremely difficult requiring the use of infectious clones for inoculation. Delayed serocon- version, slightly elevated circulating liver enzymes, and mild hepatitis was observed, followed by viral clearance. Hepatotropism was confirmed by level of viremia in the liver and by finding negative- strand RNA in the liver, a hallmark of viral replication.107 In August 2011 we were asked to consult on an outbreak of acute hepatitis in horses.110 Two and a half months earlier four horses from the affected farm had developed botulism caused by ingestion of toxin-contaminated hay. Twenty-two horses eat- ing that hay had been treated, either therapeutically or prophylactically, with Clostridium botulinum an- titoxin (equine origin). Seven of 17 horses treated with one particular lot of botulinum antitoxin devel- oped clinical and/or biochemical evidence of hepati- tis 6–8 weeks later. Hepatitis was not observed in five horses that were treated with a separate lot of botulinum antitoxin produced from different horses or in 53 farm horses that had not received antitoxin. Using high-throughput pyrosequencing, a new vi-


rus belonging to the family Flaviviridae (the family of human HCV) was identified in the serum of two clinical cases of acute hepatitis/Theiler’s disease and in the botulinum antitoxin the horses received 47 and 52 days prior to the onset of illness. The nu- cleic acid sequences of the viruses in the two hepa- titis cases and in the botulinal antitoxin were virtually identical. The virus was named Theiler’s Disease–Associated Virus (TDAV).100 A reverse transcriptase–polymerase chain reaction (RT-PCR) then was used to test the serum of horses for the newly discovered equine hepatitis–associated virus (TDAV). All 17 of the horses that received the in- criminated lot of botulinum antitoxin tested positive


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