VENEZUELAN EQUINE ENCEPHALITIS

AETIOLOGY

Classification of the causative agent

Venezuelan equine encephalomyelitis (VEE) viruses are taxonomically classified within the genus Alphavirus of the family Togaviridae (formerly the Group A arboviruses). The VEE complex of viruses includes six antigenic subtypes (I–VI) divided by antigenic variants. Within subtype I there are five antigenic variants (variants AB–F). Originally, subtypes I-A and I-B were considered to be distinct variants, but they are now considered to be identical (I-AB). Antigenic variants I-AB and I-C are associated with epizootic/epidemic activity in equids and humans. The other three variants of subtype I (I-D, I-E, and I-F) and the other five subtypes of VEE (II–VI) circulate in natural enzootic cycles. 

Resistance to physical and chemical action

Temperature:

 Thermal inactivation point (TIP) for Alphaviruses is 58°C and virion half-life is 7 hours at 37°C

pH:

 Alphavirus virions are stable in alkaline environment of pH 7–8 but inactivated quickly at acidic pH

Chemicals/Disinfectants: 

Inactivated by various common disinfectants; sensitive to organic solvents and detergents, 1% sodium hypochlorite, quaternary ammonium compounds, phenolic disinfectants, 70% ethanol, 2% glutaraldehyde, and formaldehyde, 50% ethanol for 1 hour, ultraviolet light

Survival: 

The agent is susceptible to radiant sunlight, moist or dry heat and drying; thus cool, moist, dark conditions favour survival

EPIDEMIOLOGY

Hosts

• Enzootic VEE viruses (I-D, I-E, I-F and subtypes II–VI) are primarily found cycling between 

sylvatic rodents and Culex mosquitoes (Melanoconion subgenus)

o Wild rodents are the usual reservoir hosts for enzootic VEE viruses

o The potential role for birds requires further clarification and may depend on virus strain

o Equids and humans are considered incidental or dead-end hosts 

• Natural or inter-epidemic host for epizootic strains (I-AB and I-C) is yet undetermined; prevailing science indicates that the epizootic strains emerge from genetic modification of enzootic strains

o Equidae serve as amplifying hosts for epizootic VEE virus strains; horses and donkeys produce high viraemias which, in turn, infect a wide range of mosquitoes

o Humans and occasionally other mammals infected with epizootic strains of VEEV can develop viraemia sufficient to infect mosquitoes, but are not thought to be important in the epidemiology of this disease

o During VEE epizootics, virus has been isolated from cotton rats, opossums, gray fox, bats and many wild birds

o Cattle, swine, chickens and dogs have been shown to seroconvert after epizootics; mortality has also been observed in domesticated rabbits, dogs, goats and sheep

o Experimentally, guinea-pigs, mice, hamsters and some non-human primates have been infected; laboratory rodents are usually subclinically infected but some isolates can be fatal

Transmission

• Haematophagous insects play a central role in the transmission of all VEE viruses

o Epizootic strains have been isolated from the genera: Aedes, Anopheles, Culex, Deinocerites, Mansonia and Psorophora 

o Mechanical transmission of epizootic VEE has been demonstrated for blackflies (Simulium spp.)  

o Role of ticks uncertain though Amblyomma and Hyalomma species have been experimentally infected with both enzootic and epizootic strains of VEE 

• Enzootic/endemic or Sylvatic cycle: enzootic VEE variants and subtypes cycle in tropical ecosystems among rodents, and perhaps birds, by the feeding of mosquitoes  

• Epizootic/epidemic cycle: equids are amplifying host (high, prolonged viraemias) and infect a wide range of mosquitoes which are not restricted to equid hosts 

o Non-vector spread of disease via direct contact or aerosols has been proposed; horse- to-human and human-to-human transmission has not been recorded 

o Role of non-equid vertebrates in transmission is not clear but likely minor

• Swiftness of spread depends on: VEE virus subtype, density of competent vectors, and population of susceptible hosts; large, geographically dispersed epizootics of VEE have depended on ability of virus to produce high viraemia in equids 

Sources of virus

• Equids (horses, donkeys, and zebras) are a primary source of epizootic virus strains during an outbreak 

o Inter-epidemic maintenance of epizootic strains is not known, though three hypotheses prevail: 

▪ Incomplete inactivation of subtype I-AB VEE vaccines (suggested by sequencing studies) 

▪ Epizootic/epidemic subtype I-AB and I-C VEE strains arise from genetic mutation of enzootic subtype I-D strains (supported by genetic studies) 

▪ Studies of epizootic I-C VEE strains in Venezuela would indicate persistent, low-level sylvatic maintenance in a yet unknown cycle (cryptic transmission cycle) 

• Haematophagous vectors become infected with high titres of virus from infected horses

• Enzootic/endemic strains remain in tropical ecosystems in a cycle between rodents and mosquitoes; and for some subtypes, birds also. 

Occurrence

Enzootic/endemic VEE viruses are known to be continually circulating in lowland tropical and sub-tropical forests and swamps or in areas classified as tropical wet forest, i.e. those areas with a high water table or open swampy areas with meandering sunlit streams. These are the areas of the Americas where rainfall is distributed throughout the year or areas permanently supplied with water. Epizootic VEE viruses were historically limited to South America, but have spread to Central America, Mexico, and the southern USA. However, epizootics in North America have been limited.

DIAGNOSIS

For the purposes of the Terrestrial Code, the incubation period for VEE is 5 days, and the infective period is 14 days. Clinically, the incubation period is typically 1– 5 days; high fever appears within 12–24 hours and neurologic signs can occur from approximately 5 days to 4 weeks after infection, depending on virus strain and equine species affected. 

Clinical diagnosis

Although a presumptive diagnosis of “equine encephalomyelitis” can be made when susceptible animals in tropical or subtropical areas display clinical signs of encephalomyelitis where haematophagous insects are active, VEE can only be considered one of various possible causes and final diagnosis will require laboratory confirmation.  

• Although enzootic VEE viruses tend not to cause overt signs of disease in equid hosts this may not always be the case as was seen with I-E virus in Mexico in 1993 and 1996. Enzootic VEE viruses can cause clinical disease in humans

• Epizootic virus strains can result in severe disease of horses, mules, donkeys and zebras

o Vary in their virulence

o Some cause febrile disease with no neurologic signs

• Infections, as measured by circulating antibody, can be as high as 90%, but morbidity will vary depending on strain and immune response 

o Morbidity rates can vary anywhere from 10–40% to 50–100%

o Mortality rates in horses can be 50–70%

Clinical disease can be characterised in four general presentations:

Subclinical

• No real manifestation of disease

• Most often associated with enzootic strains of VEE

Moderate

• Characterised by inappetance, pyrexia and depression

• The first sign observed with infection of epizootic VEE virus is fever within 12–24 hours; viraemia occurs concurrently with the onset of fever, typically lasts 2-5 days, but may occur for several weeks, possibly delayed or biphasic; termination of viraemia coincides with the production of neutralising antibodies 

Severe-non-fatal

• Continued anorexia and high fevers, with tachycardia and depression progressing to more severe central nervous system involvement 

o Paresis, muscle fasciculation and spasms; incoordination, staggering, inability to stand upright resulting in open stance to prevent falling 

o Blindness

o Head pressing, bruxism (grinding of the teeth), circling or rocking on limbs; paddling in animals which have fallen or are in lateral recumbency 

o Stupor and/or convulsions often resulting in permanent neurologic damage

• Some animals may demonstrate diarrhoea and colic

Fatal

• Similar signs to severe disease but concluding in death

• Death can be sudden or occur within hours from the onset of neurological signs

• Prolonged disease may result in dehydration and extreme loss of condition in animals and these may also eventually perish 

• Epizootics of VEE have also resulted in the deaths of other animals: rabbits, goats, dogs and sheep 

Lesions

• Gross lesions of the central nervous system in horses associated with VEE are, in general, nonspecific; varying from no lesions to extensive necrosis with haemorrhages. 

• Presence of ecchymotic haemorrhages may be due to self-induced ante-mortem trauma

• Lesions in other organs are too variable to be of diagnostic use

• Necrotic lesions in the pancreas, adrenal cortex, heart, liver, and vasculature walls

• Histopathologically, the predominant lesions are related to a diffuse necrotising meningo- encephalitis; varying from some perivascular mixed cellular reaction to marked vascular necrosis with associated haemorrhages, gliosis, and clear neuronal necrosis 

• Severity of lesions tend to be most severe in the cerebral cortex and become progressively less severe toward the cauda equina 

• Evidence of central nervous system lesions is directly related to severity and duration of the clinical signs 

Differential diagnosis

• Eastern or Western equine encephalomyelitis (EEE and WEE)

• Japanese encephalitis

• West Nile fever

• Leucoencephalomalacia due to mouldy corn intoxication (Fusarium spp.)

• Rabies

• Tetanus

• African horse sickness

• Bacterial meningitis

• Toxic poisoning

Laboratory diagnosis

Those who handle infectious VEE viruses or their antigens prepared from infected tissues or cell cultures should be vaccinated and shown to have demonstrable immunity in the form of VEE virus-specific neutralising antibody. Laboratory manipulations should be carried out at an appropriate biosafety and containment level determined by biorisk analysis (see Chapter 1.1.4 Biosafety and biosecurity: Standard for managing biological risk in the veterinary laboratory and animal facilities). A confirmatory diagnosis of VEE is based on the isolation and identification of the virus or on the demonstration of seroconversion. 

Samples

Identification of the agent

• Heparinised blood of febrile animals in an early stage of infection and closely associated with clinical encephalitic cases 

• Brain and piece of pancreas unfixed A complete set of tissues in 10% formalin from recently dead animals 

• It is often difficult to isolate VEE viruses from the brains of infected equids

Serological tests

• Paired sera, if the animal survives

o One sample at time of fever and convalescent phase serum sample should be collected 5–9 days after the collection of the first acute phase sample or at the time of death 

o If previously vaccinated, paired sera 2-4 weeks apart

Procedures

Identification of the agent

• Virus isolation in laboratory animals

o Inoculation of blood or sera of infected animals in 1–4-day-old mice or hamsters intracerebrally or by the inoculation of other laboratory animals (guinea-pigs and weaned mice) 

• Virus isolation in cell culture

o Inoculation of various cell cultures

o Inoculation of duck or chicken embryo fibroblasts

o Inoculation of embryonated chicken eggs

• Isolates can be identified as VEE virus by reverse transcriptase-polymerase chain reaction (RT- PCR), complement fixation (CF), haemagglutination inhibition (HI), plaque reduction neutralisation (PRN), immunofluorescence 

• VEE virus isolates can be characterised by indirect fluorescent antibody or PRN tests using monoclonal antibody or by nucleic acid sequencing. VEE virus characterisation should be carried out in a reference laboratory

Serological tests

• Diagnosis of VEE virus infection in equids requires the demonstration of specific antibodies in paired serum samples collected in the acute and convalescent phases 

o PRN antibodies appear within 5–7 days after infection

o CF antibodies within 6–9 days after infection

o HI antibodies within 6–7 days after infection

• Vaccination history must be taken into account when interpreting any of the VEE serological test results 

o In horses not recently vaccinated, demonstration of VEE-specific serum IgM antibodies In a single serum sample supports recent virus exposure. 

• Any diagnosis of VEE in an individual that is based on seroconversion in the absence of an epizootic should be made with care 

• Although enzootic subtypes and variants are non-pathogenic for equids, infection will stimulate antibody production that can cross-react on diagnostic tests with epizootic VEE virus variants

PREVENTION AND CONTROL

Sanitary prophylaxis

• Epizootics of VEE are most effectively controlled by taking action on the primary amplifiers – equids 

o Quarantine and movement controls of all Equidae

o Vaccination of equids

• Vector control measures

o Elimination of mosquito breeding locations (i.e. pooled or stagnant water)

o Stabling equids in screened housing; especially during prime daily mosquito activity

o Use of repellents and fans

Medical prophylaxis

• No specific therapy for viral encephalitides; supportive care

o Administration of fluids to horse unable to drink

o Carefully monitored application of anti-inflammatory agents

o Use of anticonvulsants in cases with central nervous system involvement

• Approved vaccines against VEE are:

Attenuated virus vaccine (made with strain TC-83)

o Should be reconstituted with physiological saline and used immediately; any vaccine not used within 4 hours of reconstitution should be safely discarded 

o Animals over 3 months of age are vaccinated subcutaneously in the cervical region with a single dose; annual revaccination is recommended 

o Immunogenic when given by intramuscular injection; may cause adverse reactions in recipient 

Inactivated virus vaccine (made with strain TC-83)

o Administered in two doses with an interval of 2–4 weeks between doses

o Annual revaccination is recommended

o Directions for use provided with commercial products should be followed

• Formalin-inactivated virulent VEE virus preparations should never be used in equids

o Residual virulent virus can remain after formalin treatment, and thereby cause severe illness in both animals and humans 

o Epizootics of VEE have been attributed to the use of such formalin-treated viruses