1. Re-emergent Threat of Equine Herpesvirus-1 Neurologic Disease Peter J. Timoney Department of Veterinary Science Gluck Equine Research Center University of Kentucky, Lexington, KY 40546-0099 Department of Veterinary Science Gluck Equine Research Center University of Kentucky, Lexington, KY 40546-0099

2. Respiratory Outcomes of EHV-1 Infection in Horses Ack. Dr. G. Allen (2008) Abortion Neonatal Death Neurological (EHM)

3. General features – Equine Rhinopneumonitis ► Contagious disease of equids endemic in vast majority of domesticated equid populations. ► Term encompasses range of syndromes caused by either EHV-1 or EHV-4. ► Of 5 herpesviruses known to infect the horse, EHV- 1 & EHV-4 are the 2 of greatest veterinary medical significance. ► Believed EHV-1 / EHV-4 have co-evolved with horses over millions of years. ► Neither virus of public health significance. 3/14

4. EHV-1 and EHV-4 Infections EHV-1Infection not only of respiratory tract epithelium and associated lymphatic glands but also vascular endothelium especially of nasal mucosa, lung, adrenal, thyroid and in the case of some strains, CNS and endometrium. EHV-4Infection restricted primarily to respiratory tract epithelium and associated lymph glands. Some strains can set up a leukocyte-associated viremia. 3/14

5. Industry Concerns ► EHV-1 best known for its economic impact as a cause of contagious abortion worldwide. ► EHM of concern not only economically but also from a welfare viewpoint because of the distressing nature of the disease. ► Lack of a commercial vaccine of proven capability to prevent EHM. 3/14

6. Equine Herpesvirus Myeloencephalopathy 1966 - First definitive association between EHV-1 and myeloencephalopathy following isolation of the virus from brain and spinal cord of a horse with severe neurologic disease. (Saxegaard, F., Nord. Vet. Med., 1966). 3/14

7. Equine Herpesvirus Myeloencephalopathy ► Syndrome recorded with increasing frequency over past 5-10 years, can be associated with high morbidity & case fatality rate. ► Usually a sequel to a primary respiratory infection, febrile episode or abortion. ► Can occur in horses of any age, breed or either gender. ► Nature of illness dependent on location & severity of lesions in CNS. ► Disease most frequently associated with infection with neuropathogenic strains of EHV-1. General features – 3/14

8. Equine Herpesvirus-1 Myeloencephalopathy ► Many outbreaks of EHM associated with venues / premises where significant numbers of horses are congregated together. ► Conditions at shows, etc, conducive to respiratory transmission of EHV-1 by direct / indirect means. 3/14

10. Increase in Incidence of Outbreaks of EHV-1 Neurologic Disease, 1970 - 2006 Time interval No. of neurologic disease outbreaks (US and UK) from which virus or viral DNA were available 1970 – 75 1976 – 80 1981 – 85 1986 – 90 1991 – 95 1996 – 2000 2001 – 2006 1970 – 75 1976 – 80 1981 – 85 1986 – 90 1991 – 95 1996 – 2000 2001 – 2006 1 3 4 6 5 6 33 1 3 4 6 5 6 33 Ack: Dr. G.P. Allen

11. Equine herpesvirus myeloencephalopathy caused by the hypervirulent, mutant (neuropathogenic) strain of the virus designated by USDA a potentially emergent disease of the horse. (USDA: APHIS: VS: CEAH: Center for Emerging Issues Information Sheet, January 2007) 3/14

12. Association of Novel Genotype of EHV-1 with Neurologic Disease ► Majority of severe and sometimes extensive EHM events associated with novel virus genotype. ► Novel genotype characterised by single point mutation on catalytic subunit of viral DNA polymerase. ► Guanine substituted for adenine at position 2254. 3/14

13. -- GTC GAC TAC -- -- GTC AAC TAC -- (neutral) Asparagine (neutral) Asparagine Aspartic acid (acidic) Aspartic acid (acidic) Replicase gene Replicase gene EHV-1 DNA Abortion Strains: Paralytic Strains: Nucleotide Substitution in Neuropathogenic Strains of EHV-1 Ack: Dr. G.P. Allen

14. Outbreaks of EHV-1 Neurologic Disease in USA, 2000 - 2006 --- Genotype of Virus Isolates --- Outbreaks of EHV-1 Neurologic Disease in USA, 2000 - 2006 --- Genotype of Virus Isolates --- 2000 – 2006 26 2 24 Time Span CNS Outbreaks Wild-Type Mutant No. of EHV-1 Wild-Type Outbreaks Mutant Outbreaks High neurologic morbidity High neurologic mortality High neurologic morbidity High neurologic mortality Low neurologic morbidity Low to zero neurologic mortality Low neurologic morbidity Low to zero neurologic mortality Ack: Dr. G.P. Allen

15. Clinical Outcome in Relation to Virus Genotype Involved ► In terms of both neurologic-attack and case-fatality rates, clinical outcome can vary depending on genotype of EHV-1. ► Outbreaks caused by G 2254 tend to be more extensive and clinically more severe. ► In comparison, A 2254 strains associated with lower neurologic-attack and case- fatality rates. 3/14

16. Characteristics of Vasculitis Equine Herpesvirus Myeloencephalopathy ► Perivascular cuffing with mononuclear cells and neutrophils. ► Extension of inflammatory cells from intima into media and adventitia of vessel wall. ► Endothelial proliferation and necrosis. ► Necrosis of media. ► Occasionally, thrombin in vessel lumen. 3/14

18. EHV-1 Paralysis Results from Endothelial Cell Infection Spinal Cord Blood Vessel of Paralyzed Horse EHV-1 infected endothelial cells Fibrin thrombus Inflammatory lymphocytes Ack: Dr. G.P. Allen

19. Neuropathogenic Strains of EHV-1 ► Most frequently but not invariably associated with a single point mutation in the catalytic subunit of the gene (ORF30) encoding the viral DNA polymerase gene. ► "Turbo-charged" versions of wild type virus. ► Total body burden of mutant strains of EHV-1 much greater than wild type virus. ► No evidence of neurotropism. Summary of properties – 3/14

21. Viremia in Foals after Inoculation with G 2254 Mutant or Wild Type Strains of EHV-1 n = 10 foals/group Days Post-Inoculation with EHV-1 Magnitude of Viremia 5 10 15 20 0 0 100 200 300 400 Mutant EHV-1 Wild Type EHV-1 Ack: Dr. G.P. Allen

22. Replicative Capacities of A 2254 and G 2254 Genotypes of EHV-1 ► A 2254 and G 2254 genotypes differ significantly in their respective replicative capacities. ► Cell-associated viremia and duration of respiratory shedding greater in cases of G 2254 infection. ► Infection with G 2254 strains results in vasculitis in the CNS that is more severe and more widespread. 3/14

23. Consequences of Mutation on Pathogenicity of Genetic Variants of EHV-1 ► Enhanced replicative capacity ► Elevated level of viremia ► More widespread vasculitis ► Greater severity of vasculitis ► Greater mortality from neurologic disease Ack: Dr. G.P. Allen

24. Clinical Outcome following Infection with Neuropathogenic Strains of EHV-1 ► Infection with G 2254 strains may not necessarily result in development of neurologic disease. ► Individual animal outcomes can be influenced by age, innate immunity, acquired immunity, challenge dose, hormonal status and environmental factors. 3/14

26. Evidence that A2254 Nucleotide Substitution not the Only Determinant of Neuropathogenicity not  Report that 24% of the isolates from horses with neurological disease possessed the A2254 and not the G2254 genotype (Perkins et al., 2009).  Identification of viruses with nonsynonymous nucleotide substitutions in ORF30 besides A2254 to G2254 from horses without signs of neurologic disease (Smith et al., 2010). Ack: Dr. U. Balasuriya (2011) (continued)

27.  Sequence analysis of EHV-1 field strains has identified other mutations outside of the small region of ORF30 sequenced by Nugent et al. (2006).  Mutations found in same gene or genes encoding proteins of viral elongation complex or viral envelope proteins. Ack: Dr. U. Balasuriya (2011) Evidence that A2254 Nucleotide Substitution not the Only Determinant of Neuropathogenicity (continued)

28. Factors Involved in the Epidemiology of Equine Herpesvirus Myeloencephalopathy ► Virus strain. ► Modes of transmission. ► Immune status of individual animals / groups of horses. ► Existence of the carrier state. ► Various management practices. 3/14

29. Ack: Dr. G.P. Allen

30. EHV-1 and EHV-4 Infections ► Latency of EHV-1, EHV-4 widespread (40-60%) in adult equids. ► Individual animals may be carriers of one or both viruses. ► Sites of latency of EHV-1 / EHV-4: lymphoreticular tissues associated with the respiratory tract, circulating CD3 + lymphocytes, and the trigeminal ganglia (EHV-1). Latency – 3/14 (continued)

31. ► Carrier state probably life-long. ► No infectious virus present unless latent virus has been reactivated. ► Latent virus can be reactivated by environmental / pharmacological stimuli. 3/14 EHV-1 and EHV-4 Infections Latency (cont.) –

32. Expansion in the Reservoir Size of the Latent G2254 Neuropathogenic EHV-1 Strains in Kentucky TB Mares Mutant Strain of EHV-1 (% of Total Isolates) Decade 1960’s 1970’s 1980’s 1990’s 2000’s n = 450 abortion isolates of EHV-1 5% 10% 15% 20% Smith, K. 2007. Master’s Thesis. University of Kentucky Ack: Dr. G.P. Allen

33. Prevalence of Latent, G2254 Neuropathogenic Strains of EHV-1 in TB Mare Population of Kentucky Sub-maxillary lymph nodes collected from 132 necropsied TB mares. Tested for latent EHV-1 DNA by PCR. 46% of tested mares harbored latent wild-type EHV-1 DNA. 8% of tested mares harbored G2254, neuropathogenic strains of EHV-1 (=18% of total latent reservoir of EHV-1). Sub-maxillary lymph nodes collected from 132 necropsied TB mares. Tested for latent EHV-1 DNA by PCR. 46% of tested mares harbored latent wild-type EHV-1 DNA. 8% of tested mares harbored G2254, neuropathogenic strains of EHV-1 (=18% of total latent reservoir of EHV-1). EHV-1 DNA in SMLN tissue of TB mares 132 TB broodmares 46% WT 8% M Ack: Dr. G.P. Allen

35. Development of Equine Herpesvirus Myeloencephalopathy ► Existing levels of cytotoxic T-lymphocyte precursor (CTLP) cells specific for EHV-1 critically important. ► Significantly greater risk in elderly horses (≥ 20 y.o.). ► Significantly greater risk in horses exposed to ORF30G 2,254 genotype of EHV-1. ► No significant correlation with pre-exposure levels of serum neutralising antibodies to EHV-1). Risk factors – (G.P. Allen, AJVR, 69:1595-1600, 2008) 3/14

36. Relationship Between EHV-1 Cellular Immunity and Viremic Load Cellular Immunity (Pre-Infection CTLp Frequency per million PBMC) Viremic Load (Log 10 ) Ack: Dr. G.P. Allen

37. Dr. Roger Doll, 1960’s

38. Equine Herpesvirus Myeloencephalopathy ► One of 5 clinical syndromes caused by EHV-1 and infrequently, certain strains of EHV-4. ► An emergent disease of increasing veterinary medical and economic significance since 2000. ► Usually a sequel to a primary herpesvirus respiratory infection, febrile illness or abortion. ► Can occur in horses of any age, breed or either gender. (continued) Key points – 2/12

39. ► Nature of illness depends on location and severity of lesions in CNS. ► Majority of outbreaks caused by hypervirulent, neuropathogenic (mutant) strains of EHV-1. ► Neuropathogenic EHV-1 strains give rise to much greater body burdens of virus than wild type virus. ► Neuropathogenic EHV-1 strains cause higher morbidity and case-fatality rates. (continued) Equine Herpesvirus Myeloencephalopathy Key points (cont.) – 2/12

40. ► Evidence of increasing prevalence of latent infection with neuropathogenic strains of EHV-1. ► Risk factors associated with development of EHM:  Age (≥ 20 years old).  Infection with neuropathogenic strain of EHV-1.  Level of CTLP cells specific for EHV-1. ► Very doubtful current vaccines effective in preventing EHM. Key points (cont.) – Equine Herpesvirus Myeloencephalopathy 2/12