Re-emergent Threat of Equine Herpesvirus-1 Neurologic Disease - Dr. Peter Timoney, Professor and Holder of the Frederick Van Lennep Chair in Equine Veterinary Science, University of Kentucky, from the 2014 NIAA Annual Conference titled 'The Precautionary Principle: How Agriculture Will Thrive', March 31 - April 2, 2014, Omaha, NE, USA. More presentations at http://www.trufflemedia.com/agmedia/conference/2014_niaa_how_animal_agriculture_will_thrive

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

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-1 Infection 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-4 Infection 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
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
Aspartic acid
(acidic)
Replicase
geneEHV-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 ---
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
• 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 G2254 tend to be
more extensive and clinically more severe.
► In comparison, A2254 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 G2254
Mutant or Wild Type Strains of EHV-1
n = 10 foals/group
Days Post-Inoculation with EHV-1
MagnitudeofViremia
5 10 15 20
0
100
200
300
400
Mutant EHV-1
Wild Type EHV-1
Ack: Dr. G.P. Allen

22. Replicative Capacities of A2254 and G2254
Genotypes of EHV-1
► A2254 and G2254 genotypes differ significantly in
their respective replicative capacities.
► Cell-associated viremia and duration of
respiratory shedding greater in cases of G2254
infection.
► Infection with G2254 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 G2254 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
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
MutantStrainofEHV-1
(%ofTotalIsolates)
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).
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
ORF30G2,254genotype 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)
ViremicLoad(Log10)
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