Equine parvovirus hepatitis

Abstract Equine parvovirus hepatitis (EqPV‐H) was first described in 2018 in a fatal case of Theiler's disease which followed the administration of an equine‐origin biological product. The virus has since been frequently identified in serum and liver tissue of horses affected by Theiler's disease—an acute, severe hepatitis characterised by fulminant hepatic necrosis with a fatal outcome in most cases. EqPV‐H is hepatotropic, appears to be associated with subclinical to severe hepatitis in horses, and is a likely cause of Theiler's disease. Although this disease is most frequently reported following the administration of equine‐origin biological products, it can also occur among in‐contact horses. Horizontal transmission may be iatrogenic, via contaminated equine‐origin biological products such as equine serum, botulism or tetanus antitoxin, and mesenchymal stem cells or by means of the oral route of infection. Other horizontal transmission routes, for example, arthropod vectors, warrant further investigation. A worldwide prevalence of EqPV‐H antibodies and DNA has been reported in asymptomatic horses. EqPV‐H‐positive horses suffering from acute, severe hepatitis have reportedly developed clinical signs including icterus, lethargy, inappetence, and neurological abnormalities and have had increased liver‐associated biochemistry parameters recorded. The most common histopathological abnormalities of the liver have been hepatocellular necrosis, collapse of the lobular architecture, and lymphocytic infiltration. Most horses infected experimentally with EqPV‐H have developed subclinical hepatitis, and close temporal associations between peak viraemia, seroconversion, and the onset of hepatitis have been observed. Based on strong evidence indicating that EqPV‐H causes hepatitis in horses, veterinarians should consider this virus an important differential diagnosis in such cases. Potential risks associated with the administration of equine‐origin biological products must be emphasised.


| EQU INE ACUTE HEPATITIS
Theiler's disease, an acute hepatitis frequently occurring several weeks after administration of a biological substance of equine origin, has also been termed serum hepatitis, acute hepatic necrosis, or serum-associated hepatitis. 1 The aetiology of Theiler's disease, the most common cause of acute, severe hepatitis and liver failure in horses, has remained unidentified for more than a century. 2 Sir Arnold Theiler described this disease for the first time in 1918 in South Africa, when approximately 2% of horses developed acute hepatic necrosis and fulminant liver failure several weeks after the administration of convalescent equine antiserum. 3 Since then, the condition of equine acute hepatitis has been reported worldwide in association with the administration of various equine-origin biological products, including equine plasma, tetanus antitoxin, botulism antitoxin, antiserum against Streptococcus equi, and allogenic stem cells. 4 -14 The condition has also been reported in horses with no history of receiving biological products and in horses that were in contact with cases of acute hepatitis. 3,14,15 Due to the infectious and contagious nature of the disease, viral aetiology was suspected despite no viral agent being successfully cultured from affected animals. 2 Due to advances in unbiased deep sequencing (next generation sequencing [NGS]) during recent years, four novel equine viruses, including two pegiviruses (Theiler's disease associated virus/pegivirus D/TDAV and equine pegivirus/pegivirus E/EPgV), a hepacivirus (equine hepacivirus/EqHV), and a parvovirus (equine parvovirus hepatitis/EqPV-H) were identified as potential aetiological agents in samples from horses. 2,13,[16][17][18] Follow-up investigations shed more light on the pathogenicity of these viruses. TDAV was identified as a possible cause of Theiler's disease in 2013, 13 and it was later shown that all TDAV-positive horses in the original study were coinfected with EqPV-H. 16 For both equine pegiviruses, it was recently demonstrated that they are not hepatotropic and not associated with hepatitis but instead cause persistent infection of bone marrow. 19 In contrast, EqHV and EqPV-H are hepatotropic and have frequently been detected in cases of mild or subclinical hepatitis. 2,20,21 EqHV infection has only been reported in three cases of severe clinical liver disease. [22][23][24] EqHV was associated with chronic, severe hepatopathy in two cases 23,24 and detected in one case of acute severe hepatitis. 22 However, two of these cases were published before the detection of EqPV-H, and no EqPV-H testing was performed. 22,24 Therefore, EqPV-H infection could be excluded in only one case of chronic severe hepatopathy. 23 Since the discovery of EqPV-H and with the exception of a single case, 15 EqPV-H has been detected in all recently reported cases of Theiler's disease. 14 -16 , 25-27 EqPV-H is, therefore, a likely cause of subclinical to severe hepatitis and presumably the causative agent of Theiler's disease. 2 This review focuses on the current knowledge pertaining to EqPV-H. The chronological order of publication of research studies and case reports pertaining to EqPV-H is illustrated in Table 1.

| VIROLOG IC AL CHAR AC TERISATION OF EQ PV-H
A novel equine parvovirus was identified in 2018 in the serum and liver of a horse that died of equine serum hepatitis in Nebraska, USA, 65 days after administration of tetanus antitoxin of equine origin. 16 The administered tetanus antitoxin also contained DNA of this parvovirus. 16 This newly identified virus belongs to the family Parvoviridae, subfamily Parvovirinae, genus Copiparvovirus, and species Ungulate copiparvovirus 6 and was assigned the name equine parvovirus hepatitis (EqPV-H). 16,28 The single-stranded DNA virus exists as episomes in the liver, and the genome comprises 5,308 nucleotides (nt), encoding two large open reading frames (ORFs). 16 The first ORF (1-1779) encodes a nonstructural (NS) protein, and the second ORF (1801-4722) encodes the structural proteins (VPs). 16 Two intergenic regions of 21 nt and 583 nt, respectively, connect the ORFs. 16 DNA folding software has predicted that the long intergenic region forms one long hairpin and several other small, hairpin-like structures. 16 Parvoviruses have a small genome that does not encode for the proteins required for viral replication. 29 Therefore, they are thought to require the replication machinery from helper viruses or from the actively dividing cells of the host. 29 Alternatively, parvoviruses can also activate and use the DNA damage-repair machinery in nondividing cells for replication. 30,31 The mechanisms by which EqPV-H is replicating in the liver-a typically quiescent organ with minimal dividing cells-warrant future investigation. 2

| PRE VALEN CE AND D IS TRIBUTI ON OF E Q P V-H
EqPV-H seemingly has a global distribution without apparent geographical restrictions. Surveillance studies in clinically healthy horse populations in the United States, China, Germany, and Austria have demonstrated DNA prevalence between 7.1% and 17% (Tables 2A and 2B) 16 , 32-36 and seroprevalence between 15% and 34.7% (Tables 2A and 2B). 16,33,35 Markedly higher DNA prevalence has been reported on farms with recently documented cases of equine serum hepatitis in the United States and Canada (Tables 2A and 2B). 15,21,25 The first report of EqPV-H-associated serum hepatitis in Europe came from Slovenia, where four horses on one farm were diagnosed with fatal Theiler's disease. 26 Liver tissue from all four horses tested PCR-positive for EqPV-H (Table 2B). 26

Phylogenetic analysis of the NS and VP proteins revealed that
EqPV-H shares <50% protein identity with its phylogenetic relatives in the genus Copiparvovirus, which infect pigs, cows, deer, sea lions, and horses. 16,32,34,38,39 To date, two further equine copiparvoviruses, namely, equine parvovirus cerebrospinal fluid (CSF) and eqcopiparvovirus, have been identified in horses and sequenced. 34,38 Additionally, two earlier reports of equine parvoviruses exist in the literature. The first report of a hepatotropic equine parvovirus dates back to 1985, when an equine parvovirus was isolated from the liver of an aborted equine foetus, following 16 cases of abortion on a farm in Canada. 40 Additionally, an equine parvovirus was linked to equine synovitis in Australia in 2014. 41 As no sequence information is available for these two parvoviruses, it is unknown whether these viruses differ from the known equine parvoviruses. EqPV-H is more closely related to porcine and bovine copiparvoviruses than to the two other equine copiparvoviruses, suggesting different evolutionary origins of the equine viruses. 16,32,34,38 According to the recent reorganisation of the family Parvoviridae and the thereby revised taxonomy based on host association, EqPV-H was assigned to the species Ungulate copiparvovirus 6. 28 Recently, all 12 publicly available complete coding sequences (CDS) of the species Ungulate copiparvovirus 6-obtained in China and the United States-were compared with the CDS of four Austrian variants and found to be closely related. 33 The genetic diversity of the NS and VP genes appears to be low among different EqPV-H variants in the United States, China, Canada, New Zealand, Italy, and Germany, indicating high conservation and low genetic variability of variants circulating globally. 16,25,32,35,37 Eleven years after the first outbreak of Theiler's disease on a farm in Canada, the same partial NS1 sequence was obtained from a horse that died of Theiler's disease 8 weeks after arrival on the farm. This suggests that highly similar EqPV-H variants were circulating on this farm for an extended period of time. 25 Based on unique nucleotide

| D IS E A S E A SSO CIATI ON
Since its discovery, EqPV-H DNA has been detected in serum and/or liver of multiple patients with acute, severe clinical hepatitis, including single cases and larger outbreaks in the United States, Canada, and Slovenia. [14][15][16][25][26][27] The disease has frequently been associated with the administration of equine-origin biological products 14 but has also been reported in patients with no history of biological product administration. 15,25,26 In horses that had contact with cases of EqPV-H-associated acute hepatitis, the prevalence of hepatitis was high (15%-27%) and significantly associated with EqPV-H infection. 15 The clinical signs most frequently observed in affected horses are icterus, lethargy, inappetence, and neurological signs such as blindness, obtundation, head pressing, and ataxia. 13 (Table 3B). 14,15,[25][26][27] The outcome in the majority of horses with severe clinical signs is fatal, ranging from sudden death to humane euthanasia within days of the appearance of clinical signs. [14][15][16]25,26 Livers have been described as atrophic, flattened, flabby, friable, and/or discoloured upon post-mortem examination. 14,26,27 The most commonly reported histopathological abnormalities include hepatocellular necrosis, collapse of the lobular architecture, and lymphocytic infiltration. [14][15][16]21,26,27 However, majority of horses that were tested for EqPV-H due to contact with affected horses or for prevalence studies did not show any obvious clinical signs. Nevertheless, subclinical hepatitis with elevated liver-associated biochemistry parameters could be detected in some (Table 3B). 13,15,25,33,36 To investigate the disease association of the virus, horses have been experimentally infected using EqPV-H-contaminated equine serum or biological products. 16  In experimentally infected horses, viraemia developed 0-6 weeks after inoculation, followed by a close temporal association between peak viraemia, seroconversion, and the onset of hepatitis approximately

| Noniatrogenic horizontal transmission
Hepatitis, ranging in severity from subclinical to fatal, has also been reported in EqPV-H-positive horses with no recent history of biological product administration and in horses that were only in contact with recipients of biological products. 15,25,32,36 EqPV

| RIS K FAC TOR S: AG E , B REED ING , AND S E A SON
The vast majority of EqPV-H surveillance has been performed in adult horse populations. A slight increase in the fraction of seropositive horses was observed in association with an increasing number of years in breeding. 35 However, it was speculated that this association could be attributed to the advanced age of the horses. 35

| PR AC TI C AL AND CLINI C AL R ELE VA N CE
Despite EqPV-H research still being in its infancy, the clinical relevance of this virus is becoming increasingly apparent. Awareness of the potentially fatal consequences and legal implications of administration of contaminated biological products is crucial for equine practitioners. EqPV-H is an important differential diagnosis that has to be considered and investigated in cases of clinical and subclinical equine hepatitis-even in the absence of a history of biological product administration. In future, a better understanding of transmission routes of the virus will enable practitioners to advise owners on the implementation of appropriate preventative protocols. Although the potential detrimental effect of EqPV-H infection-particularly persistent, subclinical infections-on athletic performance of horses remains to be thoroughly investigated, this does warrant consideration in equine athletes.

CO N FLI C T O F I NTE R E S T S
No competing interests have been declared.

AUTH O R CO NTR I B UTI O N S
Review conception was done by AS Ramsauer, M. Badenhorst, and JMV Cavalleri. The review was primary written by AS Ramsauer and M. Badenhorst, and overall supervision was provided by JMV Cavalleri. All authors commented and discussed the drafts and approved the final version.