Re-emergent Threat of Equine Herpesvirus-1 Neurologic DiseaseRe-emergent Threat of Equine Herpesvirus-1 Neurologic Disease

Peter J. TimoneyPeter J. Timoney

Department of Veterinary ScienceGluck Equine Research CenterUniversity of Kentucky, Lexington, KY 40546-0099

Department of Veterinary ScienceGluck Equine Research CenterUniversity of Kentucky, Lexington, KY 40546-0099

Respiratory

Outcomes of EHV-1 Infection in Horses

Ack. Dr. G. Allen (2008)

Abortion

Neonatal DeathNeurological

(EHM)

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

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

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

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

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

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

Increase in Incidence of Outbreaks of EHV-1 Neurologic Disease, 1970 - 2006

Increase in Incidence of Outbreaks of EHV-1 Neurologic Disease, 1970 - 2006

Time intervalTime interval

No. of neurologic disease outbreaks (US and UK) from which virus or

viral DNA were available

No. of neurologic disease outbreaks (US and UK) from which virus or

viral DNA were available

1970 – 751976 – 801981 – 851986 – 901991 – 95

1996 – 20002001 – 2006

1970 – 751976 – 801981 – 851986 – 901991 – 95

1996 – 20002001 – 2006

134656

33

134656

33

Ack: Dr. G.P. Allen

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

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

-- GTC GAC TAC ---- GTC GAC TAC --

-- GTC AAC TAC ---- GTC AAC TAC -- (neutral)Asparagine (neutral)Asparagine

Aspartic acid (acidic)Aspartic acid (acidic)

Replicase geneReplicase gene

EHV-1 DNAEHV-1 DNA

Abortion Strains:Abortion Strains:

Paralytic Strains: Paralytic Strains:

Nucleotide Substitution in Neuropathogenic Strains of EHV-1

Nucleotide Substitution in Neuropathogenic Strains of EHV-1

Ack: Dr. G.P. Allen

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 242000 – 2006 26 2 24

Time Span CNS Outbreaks Wild-Type MutantTime Span CNS Outbreaks Wild-Type MutantNo. of EHV-1No. of EHV-1

Wild-Type OutbreaksWild-Type Outbreaks Mutant OutbreaksMutant 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

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

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

EHV-1 Paralysis Results from Endothelial Cell Infection

EHV-1 Paralysis Results from Endothelial Cell Infection

Spinal Cord Blood Vessel of Paralyzed HorseSpinal Cord Blood Vessel of Paralyzed Horse

EHV-1 infected endothelial cellsEHV-1 infected endothelial cells

Fibrin thrombusFibrin thrombus

Inflammatory lymphocytesInflammatory lymphocytes

Ack: Dr. G.P. Allen

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

Viremia in Foals after Inoculation with G2254 Mutant or Wild Type Strains of EHV-1

Viremia in Foals after Inoculation with G2254 Mutant or Wild Type Strains of EHV-1

n = 10 foals/group n = 10 foals/group

Days Post-Inoculation with EHV-1Days Post-Inoculation with EHV-1

Mag

nitu

de

of

Vire

mia

Mag

nitu

de

of

Vire

mia

5 10 15 20 5 10 15 20

00

100100

200200

300300

400400

Mutant EHV-1Mutant EHV-1

Wild Type EHV-1Wild Type EHV-1

Ack: Dr. G.P. Allen

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

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

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

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)

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)

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

Ack: Dr. G.P. Allen

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)

► 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.) –

Expansion in the Reservoir Size of the Latent G2254 Neuropathogenic EHV-1 Strains in Kentucky TB MaresExpansion in the Reservoir Size of the Latent G2254

Neuropathogenic EHV-1 Strains in Kentucky TB MaresM

uta

nt S

tra

in o

f EH

V-1

(%

of T

ota

l Iso

late

s)M

uta

nt S

tra

in o

f EH

V-1

(%

of T

ota

l Iso

late

s)

DecadeDecade 1960’s 1970’s 1980’s 1990’s 2000’s 1960’s 1970’s 1980’s 1990’s 2000’s

n = 450 abortion isolates of EHV-1n = 450 abortion isolates of EHV-1

5%5%

10% 10%

15% 15%

20% 20%

Smith, K. 2007. Master’s Thesis. University of KentuckySmith, K. 2007. Master’s Thesis. University of Kentucky Ack: Dr. G.P. Allen

Prevalence of Latent, G2254 Neuropathogenic Strains of EHV-1 in TB Mare Population of Kentucky

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

EHV-1 DNA in SMLN tissue of TB mares

132 TB broodmares132 TB broodmares

46% WT

8% M8% M

Ack: Dr. G.P. Allen

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

Relationship Between EHV-1 Cellular Immunity and Viremic Load

Relationship Between EHV-1 Cellular Immunity and Viremic Load

Cellular Immunity (Pre-Infection CTLp Frequency per million PBMC)

Cellular Immunity (Pre-Infection CTLp Frequency per million PBMC)

Vire

mic

Lo

ad

(L

og 1

0)

Vire

mic

Lo

ad

(L

og 1

0)

Ack: Dr. G.P. Allen

Dr. Roger Doll, 1960’s

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

► 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

► 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