Equid gammaherpesvirus 2 and equid gammaherpesvirus 5 infections

Synonyms: Equid gammaherpesvirus infections, slowly cytopathic equid herpesvirus infections (formerly equid herpesvirus 2 and equid herpesvirus 5)

GENERAL INTRODUCTION: HERPESVIRIDAE

Introduction

Equid gammaherpesviruses are a genetically heterogeneous viral subfamily characterized by a narrow host range, slow replication, lymphotropism, and widespread distribution in horse populations. Their interactions with their host are not fully elucidated and have been the subject of continued interest and research. Importantly, their widespread distribution and obscure pathogenicity has been a significant challenge in establishing an association with specific clinical and pathological conditions and in defining their significance from a diagnostic standpoint.

The prototype LK strain of equid gammaherpesvirus 2 (EHV-2), the first gammaherpesvirus to be recovered from the horse, was isolated in 1962 from the respiratory mucus of a foal with ‘catarrh and coughing’.⁸⁰Since then, a great number of equid gammaherpesvirus isolates have been recovered from horses throughout the world.^{24, 27, 41, 48, 51, 56, 58, 90, 98, 101, 121} Twenty-five years elapsed before a subset of this large virus collection was recognized as sufficiently different to be classified as a second equid gammaherpesvirus type, equid gammaherpesvirus 5 (EHV-5).^{2, 20} To date, EHV-2 and EHV-5-associated infections are ubiquitous in horses around the globe. Characterized by extensive intratypic heterogeneity (both genetic and antigenic), a long replicative cycle, slow cell-to-cell viral infectivity, and low extracellular titres of infectious virus, the equid gammaherpesviruses comprise a complex and technically challenging group of herpesviruses for both equine virologists and practitioners.^{2, 3, 19, 21, 98}

A large proportion of the world’s horse population carries gammaherpesviruses as a life-long latent infection of circulating B lymphocytes as well as a fluctuating, persistent and productive infection of epithelial cells of the nasopharynx, conjunctiva and cornea.^{16, 21, 32, 40, 56, 64, 65, 90, 91, 110, 112, 121} These viruses are intermittently shed from these mucosal surfaces of infected horses, and infected mares effectively transmit the infection to foals soon after birth, even in the presence of colostrum-derived antibodies.^{12, 19, 45, 48, 70, 108, 121} Although of low inherent pathogenicity, EHV-2 has been associated with several significant, clinically overt equine diseases, including syndromes involving the of upper and lower respiratory tracts of foals, keratoconjunctivitis, chronic follicular pharyngitis, and malaise and poor performance syndrome in young performing horses.^{6, 14, 25, 26, 29, 37, 45, 48, 51, 55, 74, 75, 91, 99, 100, 102, 106, 107} In the case of EHV-5, this equid gammaherpesvirus has been associated with a specific condition known as equine multinodular pulmonary fibrosis (EMPF).^{122, 124}

Aetiology

Upon their original identification, EHV-2 and EHV-5 were recognized as ‘slowly cytopathic orphan herpesviruses’ of the domestic horse (Equus caballus)^{52, 72} and initially classified as equine cytomegalovirus-like betaherpesviruses within the subfamily Betaherpesvirinae.^{21, 41, 52, 94, 98, 115, 119} Equid gammaherpesvirus 2  and EHV-5 were indistinguishable from each other until the advent of restriction fragment length polymorphism analysis.¹⁰⁴ Subsequent sequence analysis of their genomic DNA led to their reclassification within the subfamily Gammaherpesvirinae, genus Rhadinovirus.^{3, 104, 116} Since 2008, both EHV-2 and EHV-5 have been classified within the subfamily Gammaherpesvirinae, genus Percavirus³¹ (Figure 1) along with other gammaherpesviruses recognized in cats, ferrets, seals and bats. The two gammaherpesviruses have been historically distinguished by differences in electrophoretic mobilities, DNA restriction endonuclease cleavage patterns, titres of homologous versus heterologous antisera for neutralizing the two viruses, and Southern blotting. Currently, these techniques have been replaced by next generation sequencing of full-length genomes, which provides unparalleled superiority for the characterization of equid gammaherpesviral genomes.

The equid gammaherpesviruses have typical herpesvirus morphology and virion architecture.⁸⁰ The 184 kbp double-stranded, linear DNA genome of EHV-2 has a G+C content of 57.5 per cent and a buoyant density of 1,717 g/cm.^{3, 6, 77, 119} It is a non-isomerizing genome that comprises a large (149 kbp), central unique sequence flanked at both ends by long (17.5 and 18.3 kbp), direct terminal repeats.^{22, 105, 120}The unique region of EHV-2 also contains an unrelated pair of internal, short inverted repeats at separate locations.¹⁰⁵ By contrast, the EHV-5 genome (52 per cent G + C content) is 182 kbp in size and lacks both the terminal and internal sequence repeats.^{2, 120} A striking feature of the EHV-2 genome is that nearly a third of its DNA sequence, including the large direct terminal repeat, appears not to encode protein.¹⁰⁵ Based on recent genome sequences¹²⁰ available, the genome of EHV-2 has an estimated 78 open reading frames (ORF) encoding at least 77 distinct viral proteins.^{88, 105}Comparatively, the EHV-5 genome has an estimated 79 ORFs. EHV-2 and EHV-5 are distinct, but closely related, equid gammaherpesviruses, sharing an overall 60 per cent amino acid sequence identity among various viral protiens.^{3, 49, 97, 104} The proteins and glycoproteins of both EHV-2 and EHV-5 have been characterized.¹Many of the EHV-2 and EHV-5 structural proteins co-migrate in SDS-PAGE gels and contain both type-common and type-specific epitopes.¹ The proteins of the two viruses cross-react strongly in ELISA, Western blot, and radioimmunoprecipitation, but less so in infectivity neutralization assays.¹Equid gammaherpesvirus 2  and EHV-5 are genetically colinear with one another and closely related to human gammaherpesvirus 4 (Epstein-Barr herpesvirus), saimiriine herpesvirus 2, and, presumably, gammaherpesviruses of other domesticated livestock (e.g., alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2; causes of malignant catarrhal fever).^{49, 104, 105} Equid gammaherpesvirus 2  and EHV-5 are genetically and antigenically distant from the three alphaherpesviruses (EHV-1, EHV-3, and EHV-4) of horses.^{21, 41, 56, 77, 79, 82, 95, 98} No cross-neutralization has been reported between the equid gammaherpesviruses (EHV-2 and EHV-5) and the alphaherpesviruses EHV-1, EHV-3, or EHV-4.^{3, 79}

In cell culture, EHV-2 and EHV-5 exhibit a relatively broad host range and a long replication cycle.^{48, 55, 58, 77, 80, 98, 119} Because much of the viral infectivity remains avidly cell-associated, the spread of infection and cytopathic effect through inoculated cell monolayers is generally slow and the extracellular yield of infectious virus is low (10³ to 10⁵ TCID₅₀/ml). Typical herpesvirus intranuclear inclusion bodies with prominent clear halos are produced in cell cultures.¹¹⁰Susceptible cell lines include foetal equine kidney (FEK), primary rabbit kidney, primary feline kidney, and, in the author’s experience, equine pulmonary artery endothelial cells and the continuous rabbit kidney cell line, RK-13.

Isolates of EHV-2 from individual horses comprise a genetically, antigenically, and biologically heterogeneous group of viruses.^{1, 20, 23, 48, 77, 79, 101, 110, 111}Different EHV-2 isolates exhibit intratypic variability in their DNA restriction endonuclease fragment profiles, growth rate in cell culture, and the extent of neutralization by reference antisera. An initial study assessing nucleotide variation in the gB glycoprotein gene sequence shed by foals in nasal secretions demonstrated notable heterogeneity in EHV-2 nucleotide and deduced amino acid sequences as foals became older.¹² Whether EHV-2 isolates exhibit variability in their virulence is a clinically important but unanswered question. In spite of this genomic heterogeneity, characterization of EHV-2 strains using a panel of neutralizing monoclonal antibodies distinguished two distinct antigenic groups, namely EHV2.86/67-like and EHV2.141-like, based on reference virus strains⁵⁰. Genomic variability among isolates of EHV-5 has also been documented, whereby isolates from different horses showed a high level of heterogeneity while isolates from individual animals remained conserved when examined over a period of time.^{12, 33}The genomic heterogeneity of these equid gammaherpesviruses likely explains why horses can become infected with different strains during their life.

Epidemiology

The geographical distribution of the equid gammaherpesviruses (EHV-2 and EHV-5) is worldwide, and the infection rate is high and considered to be endemic around the globe. Equid gammaherpesvirus 2  has been isolated from the blood leukocytes of 89 per cent of horses tested in the United States of America (USA),⁵⁶ 86 per cent in Scotland,⁹⁰⁶⁴ 90 per cent in England,⁹¹ 55 per cent in Japan,⁵⁸ and 77 per cent in Switzerland.⁹¹ In Australia and the United Kingdom (UK), 97 per cent of examined abattoir horses harboured the virus in their lymphocytes.^{40, 48} The virus has also been recovered from nasal secretions.³⁰ Seroprevalence is, therefore, high.^{9, 12, 16, 30, 64, 73, 92, 109} In older horses, seropositivity for EHV-2 approaches 100 per cent.^{9, 16, 27, 64, 91} In Australia, gammaherpesvirus type-specific PCR assays were used to evaluate individual EHV-2 and EHV-5 infections in racehorses and breeding mares and showed that 31 per cent of the horses were positive for EHV-2, 16 per cent were positive for EHV-5, and 8 per cent were positive for both viruses.⁸⁴ Similar relative proportions of horses latently infected with EHV-2, EHV-5, or both EHV-2 and EHV-5 have been reported in adult horses in Switzerland.⁴³

Equid gammaherpesvirus 5  was first isolated in Australia from the nasal secretions of two quarantined horses that originated from the UK.¹¹⁰ Subsequently, this virus was identified in buffy coat samples from mares with upper respiratory tract disease,¹²¹ as well as in both healthy and clinically affected horses in the USA, Australia, and Europe.^{3, 12, 16, 34, 42, 43, 74, 84, 109, 117}

Equid gammaherpesviruses have a high rate of infection in very young foals, low morbidity, and life-long latent infection with sporadic reactivation during which infectious virus is shed into the environment.^{19, 21, 24, 27, 36, 41, 46, 48, 56, 64, 70, 90, 91, 98, 101, 110, 111, 121} Several studies have shown that EHV-2 and EHV-5 viral loads tend to be higher in juvenile compared to adult horses.^{8, 53, 61} Infection occurs in neonatal to early juvenile foals, even in the presence of high levels of colostral-derived antibodies.¹² Interestingly, EHV-2 infection occurs earlier in life than EHV-5. A significant property of EHV-2 is its capacity for persistent, intermittent or constant, shedding from the respiratory tract of the horse.^{14, 19, 48, 121} Intermittent viral shedding into the nasal mucus for up to 14 weeks has been demonstrated.^{14, 19} These results suggest that, following natural infection or reactivation of latent infection, EHV-2 can maintain a fluctuating, low-grade, productive infection of epithelial cells in the nasopharynx for a long period. The prowess of EHV-2 as a successfully adapted equine virus is substantiated by seroepidemiological and virological studies that indicate almost universal infection in young foals. It is believed that latently infected horses serve as the reservoir for transmission of infection, and clearly the equid gammaherpesviruses have adapted very well to their natural host, guaranteeing their maintenance in the equine populations.

Figure 1 Current classification of equid gammaherpesviruses based on the International Committee on Taxonomy of Viruses (ICTV) release of 2020

The precise mechanisms of equine gammaherpesvirus transmission between horses arenot understood, but the detection in nasal secretions supports the respiratory tract as the site of entry and shedding. Gammaherpesviruses periodically reactivate, during which the horse usually remains asymptomatic.^{16, 19, 40} Investigations of the dynamics of generation-to-generation transmission of the equid gammaherpesviruses have shown that, in foals as early as four to six weeks of age (i.e., within approximately the first month of life), the virus can be isolated from nasal swabs and peripheral blood leukocytes.^{45, 48, 70, 121} In three classic epidemiologic studies on a collective group of 39 mares and their nursing foals monitored over periods of six months after foaling, it was demonstrated that EHV-2 and EHV-5 were excreted into the nasal secretions of 85 per cent of foals and 40 per cent of mares.^{45, 48, 121}A more recent study by Bell et al. yielded similar results.¹² In one of the studies in which conjunctival swabs were collected, virus was also recovered from the ocular secretions of both mares and foals.⁴⁸ Most of the animals shed EHV-2 from one or both of these sites on more than one occasion during the six-month period. In one study, EHV-2 shedding reached a peak at three to four months of age.⁴⁵ The earliest ages for recovery of EHV-2 in the three studies were 25, 30, and 33 days, respectively. More recent virological monitoring for the presence of gammaherpesviruses in blood leukocytes of foals on breeding farms confirmed that by six to eight months of age virtually all foals become infected with the virus, with infection occurring as early as 25 days after birth.^{12, 70} In most cases, EHV-2 infection was not accompanied by detectable clinical signs. In other instances, however, respiratory disease characterized by nasal discharge, fever, and swollen submandibular lymph nodes was observed in foals around the time when the virus was first isolated.⁴⁵Infection of foals may take place in the presence of passively acquired maternal antibodies and is accompanied by an increase in virus-neutralizing antibody titres to EHV-2.^{12, 45, 108, 121} However, maternal antibodies affect viral load, and both EHV-2 and EHV-5 titres peak sooner in those foals with lower levels of colostral antibodies.¹⁰⁸ Prenatal infection with EHV-2 has not been recorded, and the virus has not been detected in colostrum or milk. These results suggest that the equid gammaherpesviruses are readily available in the young foal’s environment, probably originating from respiratory and/or ocular secretions of virus shedding cohorts.

The pattern of EHV-2 transmission is complex.¹⁹ Re-emergence of the latent virus in mares, accompanied by respiratory and/or ocular shedding in the presence of antibody, has been frequently recognized.^{45, 48, 121} Multiple genetic clades of virus may co-circulate within an isolated and commingling group of horses, suggesting frequent shedding and cross-infection. ¹⁹ As many as five genetically distinguishable variants of EHV-2 have been isolated from a single young horse during the course of a year of virological monitoring. ¹⁹ Concurrent infection of the respiratory tract of horses with two or more EHV-2 genotypes has been also demonstrated. ¹⁹ Reports of the isolation of genetically identical viruses from the respiratory tract of a horse over the course of many weeks indicate the potential of EHV-2 for establishing a persistent infection with sustained shedding of a single genotype of virus. ¹⁹ However, the intermittent, chronic shedding of different strains of EHV-2, either from re-infection or reactivation, is more common than persistent infection by a single strain. ^{19, 111} Evidence suggests that both vertical (mare-to-foal) and horizontal (foal-to-foal) spread of EHV-2 infection may occur.¹⁹

Pathogenesis, pathology and clinical signs

The majority of EHV-2 and EHV-5 infections remain clinically minimal or inapparent.^{27, 41, 48, 56, 70, 90, 91, 101, 110, 121} Caution must be exercised when interpreting clinical disease with the detection of EHV-2 or EHV-5 in samples from clinically affected horses, because causal relationships with specific disease entities are thus far vastly undetermined. Isolation of EHV-2 is common from nasal swabs collected from horses that lack respiratory disease and, likewise, from ocular swabs from horses in which clinically overt ophthalmic disease is not present.^{48, 60, 70, 121} Both seroconversion or an increase in existing antibody titres in foals with no clinical signs of disease have been frequently observed. ^{27, 70, 91, 110}Studies designed to investigate the pathogenicity of gammaherpesviruses in the horse are still few in number despite the work performed in recent years. Additionally, detailed pathologic analyses are not yet available following infection by the viruses, and their precise relationship to any equine disease has not been described. Nonetheless, the temporal correlation of gammaherpesviruses with several clinical entities has raised the possibility of their role as low to moderately pathogenic opportunists with the ability to cause, or to serve as co-factors in the cause of, clinically detectable disease. It is noteworthy that an association between EHV-5 infection and the development of equine multinodular pulmonary fibrosis (EMPF) has been established and reproduced experimentally in the past few years.^{122, 123, 124} This constitutes the sole, unequivocal evidence of a causal relationship between an equine gammaherpesvirus and occurrence of disease (see below).  

Equid gammaherpesvirus 2

Equine diseases that have been associated with EHV-2 infections include keratoconjunctivitis (Figure 2), both upper and lower respiratory tract diseases in foals, chronic follicular pharyngitis, and a ‘malaise and poor performance syndrome’ in young performing horses.^{14, 29, 35, 57, 59, 67, 74, 92, 98, 107} Of these, keratoconjunctivitis, chronic follicular pharyngitis, and a mild rhinitis have been reproduced by experimental inoculation of young horses with EHV-2.^{14, 17, 47} It should be remembered, in each of these proposed affiliations between EHV-2 and clinical disease, that following its initial transmission to foals at a very young age, EHV-2 becomes widely disseminated as a latent infection of leukocytes and, for the remaining lifetime of that individual animal, whether clinically ill or healthy, can be isolated with ease and great frequency from any leukocyte-containing tissues or secretions.

The best evidence for establishing EHV-2 as the causative agent of significant infectious disease of the horse derives from numerous reports of its isolation from outbreaks of keratoconjunctivitis in suckling or weanling foals.^{17, 29, 37, 57, 66, 97, 106, 107}The condition is characterized by uni- or bilateral keratitis and/or conjunctivitis and may be accompanied by excessive lacrimation, superficial corneal opacities, corneal ulcerations, supraorbital oedema, and corneal limbal neovascularization. Pyrexia, nasal discharge, cough, and photophobia are usually absent, and  compromised  vision has not been reported as a sequela. Keratoconjunctivitis may be associated with previous or concurrent respiratory tract disease in foals. The syndrome can be highly contagious, with as many as half of the cohort exhibiting ocular disease. Equid gammaherpesvirus 2  isolates recovered from the eyes of affected foals within the group may be genetically dissimilar.²⁹ A similar clinical condition has been experimentally reproduced in dexamethasone-treated yearling ponies following intranasal inoculation with EHV-2.¹⁷

Several observations have suggested a possible aetiological role of EHV-2 in upper respiratory tract disease in young foals.^{6, 14, 25, 27, 45, 51, 54, 80, 87, 91, 99, 100}The syndrome’s clinical presentation has generally been characterized as a mild ‘cold’ with excessive bilateral discharge of clear mucus from the nostrils of suckling foals, with only occasional  pyrexia or cough noted. Associated rhinitis in foals tends to cluster on individual farms with annual recurrences. The disease has a high morbidity, and its course is not influenced by the administration of antibiotics. In the absence of complications or secondary bacterial infection, clinical recovery usually occurs after a week, but chronic infections have also been noted in some foals. Pharyngeal endoscopy of affected foals  revealed, in some animals, chronic ulcers with lymphoid follicular hyperplasia, and similar pharyngitis has been reproduced by experimental, intranasal inoculation of foals with EHV-2.¹⁵ In the majority of cases, associations of EHV-2 with disease have been based on isolation of slowly cytopathic herpesviruses, with the exclusion of all other known respiratory viral pathogens of horses, from upper respiratory tract mucus samples collected by swabbing the anterior nasal passages of clinically affected foals. Despite these observations, the clinical significance of the presence of slowly growing herpesviruses in the upper respiratory tract is difficult to assess, as these viruses are commonly recovered from such locations in clinically normal foals.

Limited data supports the scenario that infective EHV-2 is aerosolized and excreted from the respiratory tract of a shedding horse. The virus then enters the new host through the upper respiratory tract where it infects and replicates in the respiratory mucosal epithelium.^{19, 48, 70, 98, 110, 121} The epithelia of the conjunctiva and cornea are also susceptible to EHV-2 infection.^{29, 37, 45, 106} Intranuclear inclusion bodies have been demonstrated in exfoliated nasal and conjunctival epithelial cells of horses from which slow-growing herpesviruses were isolated.⁸⁷ Infection of respiratory and conjunctival epithelia is productive, and infectious virus is shed into the secretions from the respiratory tract and eyes.^{29, 45, 48, 121} Equid gammaherpesvirus 2  infection of the respiratory mucosal epithelium is accompanied by infiltration of infected leukocytes within the subepithelial lymphoid tissue, the pharyngeal lymphoid follicles, or draining lymph nodes. Infected leukocytes then enter the vascular circulation. The resultant cell-associated viraemia and spread via circulating leukocytes disperses EHV-2 throughout the infected horse and results in the possibility of isolating infectious virus from many tissues. Equid gammaherpesvirus 2  infection of leukocytes is non-productive, since infectious virus is not retrieved without prior in vitro co-cultivation of the harvested leukocytes with fully permissive cells.³² Freeze-thawing or sonication of equine leukocytes prior to inoculation onto the permissive cell monolayer abrogates the recovery of infectious virus.³² The nature of the abortive infection of leukocytes by EHV-2 is unknown. Genomic viral material can be detected by PCR and by co-cultivation in the peripheral blood leukocytes of horses shortly after intranasal infection and likely remains for the life of the horse.^{3, 17, 40, 69, 84} The nature and kinetics between post-infection, cell-associated viraemia (with a lytic programme of viral gene expression) and long-term, latently infected leukocytes (with a latent programme of viral gene expression), and whether these two types of EHV-2 infected leukocytes coexist temporally, are unknown.

Circulating leukocytes and lymph nodes draining the respiratory tract are the main anatomic reservoirs for latent EHV-2.^{2, 40, 46, 86, 118} The frequency of peripheral blood mononuclear cells latently infected with EHV-2 may range between 0.5 and 25 per 10⁶ cells.⁴⁷ The specific lymphoid cell that harbours latent EHV-2 is the B lymphocyte.³² The frequency of EHV-2 infected B lymphocytes also varies considerably among horses and has been reported to range between 4 and 780 per 10⁶ cells.³² Experimentally induced reactivation of latent EHV-2, accompanied by nasal shedding of infectious virus, has been demonstrated in ponies after administration of dexamethasone.¹⁷ Endogenously reactivated EHV-2 that infects the respiratory mucosal epithelium and results in shedding of infectious virus into the respiratory secretions is thought to originate from virus reactivated in latently infected B lymphocytes during their normal circulation through, or inflammatory recruitment into, the mucosa of the equine respiratory tract. As a result of environmental or social stress factors, intermittent reactivation events in the respiratory mucosal lymphocytes, with bursts of productive infection of respiratory epithelium, apparently result in excretion and environmental dissemination of infectious virus. The transfer of reactivated EHV-2 from infected B-lymphocytes, which do not produce infectious free virions, to the respiratory epithelium requires cell-to-cell contact.³²Latent EHV-2 DNA has also been detected, albeit with less frequency (10 to 50 per cent), in the trigeminal ganglia of horses.^{40, 86}

It has been postulated that EHV-2 may play an inciting or predisposing role in a lower respiratory disease complex of young foals that is of far greater clinical and economic significance than rhinitis, and clinically known as equine acute respiratory distress syndrome (ARDS).^{6, 11, 25, 26, 54, 75, 81, 87, 99, 100, 102} The complex is characterized clinically by fever, nasal discharge, cough, and abnormal lung sounds; endoscopically by bronchial erythema and mucopurulent exudate; and pathologically by tracheobronchitis, bronchiolitis, and interstitial or bronchopneumonia. Later stages are associated with bacterial infections (e.g., Streptococcus equi or Rhodococcus equi), but several findings support the hypothesis that EHV-2 infection may be an initial, predisposing or augmenting factor for disease development. Murray et al.  isolated slowly cytopathic herpesviruses from lower respiratory tract secretions (transtracheal aspirates) of 20 out of 30 foals with clinically apparent distal respiratory tract infection, which further implies the importance of gammaherpesviruses in this respiratory disease complex of foals.⁷⁰ In Hungarian and Swedish studs, annual outbreaks of endemic rhodococcal pneumonia in foals have been controlled by active immunization with a vaccine containing EHV-2 alone⁷⁴ or in combination with R. equi.¹¹⁴ In other studies, endemic rhodococcal pneumonia has been curtailed by passive immunization with hyperimmune anti-EHV-2 serum.¹¹

Another condition of the horse that has been attributed to infection by EHV-2 is a ‘general malaise and poor performance’ syndrome of young performing animals in training or on the racing circuit.^{14, 91, 99, 100, 101}This is characterized clinically by general malaise, chronic lymphoid follicular hyperplasia, and swelling of the regional lymph nodes of the head. The primary complaint is usually that of athletic performance below the expectation of the horse’s trainers. Rises in body temperature, inappetence, nasal discharge, and cough are variably present. The syndrome is usually persistent, does not respond to antibiotic therapy, and mostly affects younger horses. The condition in horses is comparatively reminiscent of a gammaherpesvirus (Epstein-Barr virus)-induced disease in young humans (infectious mononucleosis or glandular fever).⁷²

In a recent study, in situ hybridization and nested PCR determined that both EHV-2 and EHV-5 co-infected the gastric mucosal epithelium in a small subset of horses. These gammaherpesviruses were identified regardless of the presence or absence of gastric ulcers and, therefore, an association or causal relationship between these infections and the occurrence of equine gastric ulcer syndrome (EGUS) could not be established.⁷⁶ Since these viruses induce  cell-associated viraemia and spread throughout the body, their detection in the gastric mucosa may not represent an unexpected finding. 

For a virus to cause persistent infection, the infected cell must acquire the potential to evade recognition and elimination by the host’s immune system. The genome of EHV-2 contains genes encoding potential immunomodulatory proteins with significant amino acid sequence homology to human (76.4 per cent homology) and mouse (68.5 per cent homology) interleukin-10, Epstein-Barr virus protein BCRF1 (70.6 per cent homology), and to several chemokine receptor proteins.^{28, 88, 89} The functional ability of these virus-encoded proteins to counteract or subvert the local immune defence mechanisms of the horse may, in addition to enabling persistent EHV-2 infection, augment or exacerbate infection by other microbial pathogens. Observations have been reported on the suppression of lymphocyte activity, both in vitro and in a rabbit model, after infection by EHV-2.^{5, 78} Such an immunosuppressive role for EHV-2 has been indicated by reports of the virus acting as a co-factor in the infection of young foals with R. equi.⁷⁴

It has been suggested that EHV-2 may play a transactivating role in the reactivation of latent alphaherpesviruses of the horse, as the growth of EHV-2 during co-cultivation studies always accompanies the recovery of latent EHV-1 and EHV-4 from lymphoid tissue.¹¹⁸

The recent, successful transmission of EHV-2 respiratory tract infection to the mouse should provide a useful laboratory animal infection model for future studies on EHV-2 pathogenesis.⁸⁵

Figure 2 Viral inclusion bodies (arrowheads) in corneal epithelial cells from a corneal scraping derived from a foal with keratoconjunctivitis and positive for EHV-2 by real-time qPCR. Affected corneal epithelial cells are characterized by margination of the chromatin, dissolution of the nuclear membrane, cell swelling and also show evidence of syncytia formation. Image kindly provided by Dr. Melissa Swan, , University of Kentucky Veterinary Diagnostic Laboratory, Lexington, KY, USA.

Equid gammaherpesvirus 5^{122, 123, 124}

In 2007 and 2008, EHV-5 was unequivocally determined to be the cause of a sporadic clinical condition in  horses known as equine multinodular pulmonary fibrosis (EMPF). This entity is a chronic, progressive and interstitial lung disease observed in adult horses, with poor prognosis. As its name indicates, it is grossly featured by multiple to coalescing nodules that replace the pulmonary parenchyma. Nodules are characterized microscopically by areas of prominent interstitial fibrosis, mononuclear to mixed interstitial inflammation, and air spaces filled with macrophages and cell debris and lined by prominent, hyperplastic type II pneumocytes (Figures 3 and 4). Occasionally, intranuclear, eosinophilic viral inclusion bodies are evident within luminal macrophages. Importantly, not all horses infected with EHV-5 develop EMPF, and other factors likely play a role in the development of this condition. The combination of gross and histopathology is the gold-standard for the diagnosis of EMPF, and positive PCR results alone are not  confirmatory. A recent study evaluating EHV-5 DNA within bronchoalveolar lavage samples demonstrated that EHV-5 could be detected in 91 per cent of horses with EMPF and PCR, in addition to other complementary methods,⁸³ could be used to aid in the ante-mortem diagnosis of EMPF. Other disorders to which EHV-5 has been associated include lymphoproliferative disorders, dermatitis, systemic granulomatous disease and ocular disease,^{10, 113} some of which are reminiscent of human diseases induced by gammaherpesviruses, such as Epstein Barr virus and Kaposi-sarcoma-associated herpesvirus. However, no other disease has been strongly associated with EHV-5 infection, and the frequency of EMFP is rare despite the high rate of EHV-5 detection in the horse population.

Induction of EMPF following EHV-5 infection was experimentally attempted to further understand the pathogenesis. Of six animals used in the study, only 50 per cent developed gross lesions while histological changes were noted in the majority of animals (5/6). As hypothesized from the pattern of lesion development in natural cases, the lesions worsened as the study progressed. Unexpectedly, virus was not recovered from affected tissues and bronchoalveolar lavage samples. This challenge model encountered significant challenges due to the high prevalence of infection in healthy animals and the significant time needed for chronic lesions to develop. 

Most recently, studies have focused on unravelling virus-host interactions occurring early after infection using ex vivo and in vitro culture models, and characterizing the cellular tropism during latency.^{65, 112} While EHV-5 was unable to infect respiratory epithelial cells, it successfully infected low numbers of pneumocytes in lung explants. This study also determined that monocytes do not support viral replication, while the virus replicates at a low level (in up to 10 per cent) of  T and B lymphocytes. Based on this study, the hypothetical pathogenic model of EHV-5 infection includes initial infection of lymphocytes (B and T) residing in the tonsils, with subsequent spread into the bloodstream and draining lymph nodes, where additional lymphocytes become infected. Infected lymphocytes are suspected to re-route and home along the respiratory tract with transfer and shedding of virus particles through the mucosal epithelial lining. In combination with an additional study, it was determined that EHV-5 preferably establishes latency in B lymphocytes. In the lung, EHV-5 would have the capacity to infect pneumocytes and trigger host inflammatory responses leading to the development of interstitial fibrosis.

Figure 3 (A and B) Gross lesions of equine multinodular pulmonary fibrosis (EMPF) associated with EHV-5 infection. The pulmonary parenchyma is multifocally replaced by discrete, white and firm nodules that correspond to areas of pulmonary interstitial fibrosis.

Figure 4 (A-D) Microscopic alterations of equine multinodular pulmonary fibrosis (EMPF). The grossly visible nodules are microscopically characterized by pronounced interstitial fibrosis, mononuclear interstitial inflammation, type II pneumocyte hyperplasia, and air spaces filled with macrophages and karyorrhectic cellular debris. Rare intranuclear inclusion bodies can be identified (D, arrow).

Immune response to equid gammaherpesviruses

Studies evaluating the immune response following EHV-2 and EHV-5 infections are limited. As indicated above, the level of EHV-2 shedding in foals decreases when antibody levels raise. However, antibody titres do not correlate with protective immunity.^{16, 57, 62} A few studies demonstrated a significant interferon gamma response similar to infection with EHV-1.^{18, 103}

Diagnosis

Determination of the significance of EHV-2 and EHV-5 infection is complicated by the ubiquity of gammaherpesviruses within horse populations and by their natural propensity for establishing persistent infection with chronic shedding, as well as a lifelong latent infection. The mere demonstration, therefore, of the presence of either infectious virus or viral DNA within clinical specimens collected from the horse does not differentiate between primary-active, secondary-active (i.e., reinfection), persistent, and latent infections by EHV-2/EHV-5. Furthermore, recovery of a gammaherpesvirus from a horse exhibiting clinical disease does not, by itself, establish a direct causal relationship with disease. Currently, both EHV-2 and EHV-5 have been detected in both healthy and sick horses.

Those caveats having been stated, the presence of EHV-2/ EHV-5 in tissues or secretions of a horse can be detected by:

• isolation of the viruses in cell culture, or
• by polymerase chain reaction (PCR).

Samples to be submitted to the laboratory for detection of EHV-2/EHV-5 infection should include respiratory tract secretions obtained by nasopharyngeal swab or by transtracheal wash, 20 ml of heparinized blood for leukocyte isolation, a clotted blood sample for serum, and, if clinically indicated, secretions collected from the eyes of animals suffering from ophthalmic disease.

The laboratory isolation of infectious gammaherpesvirus (EHV-2 and EHV-5) from swabs, secretions, or by co-cultivation from the leukocyte fraction of blood or tracheal aspirates can be achieved by their inoculation onto monolayers of permissive cells [e.g., foetal equine kidney (FEK), primary rabbit kidney, primary feline kidney, equine pulmonary artery endothelial cells, or a continuous rabbit kidney cell line (RK-13)]. Of these, FEK cells are most sensitive for primary isolation.⁶⁹ Co-culture ratios may influence the success rate of virus isolation, and a previous study has determined that high co-culture ratios may actually reduce success rates.⁹⁰

Equine kidney cell cultures prepared from adult horses should not be used for laboratory isolation of the gammaherpesviruses, as endogenous contamination of such cell cultures by slow-growing herpesviruses has been reported.^{41, 52, 55, 58} There is no evidence for the presence of endogenous EHV-2/EHV-5 in cell cultures prepared from foetal equine kidneys.

In cell culture, equine gammaherpesvirus cytopathology (CPE) is distinctly focal in appearance. The foci of rounded, refractile cells are usually observed between five and fourteen days after primary inoculation and between three and eight days on subsequent passage (Figure 5). During the first three to four passages, transfer of the virus with the cell-free supernatant of inoculated cultures exhibiting CPE may not be successful.⁷⁵ In some instances, evidence of infection by EHV-2 may not be observed, or may be difficult to detect, after primary inoculation of clinical material into cell cultures, but will appear after blind passage of an aliquot of the contents of the culture flask (both cells and medium).^{56, 90, 91, 121} As many as four blind passages may be required before the appearance of gammaherpesviral CPE.⁹¹ Epidemiologically distinct isolates of EHV-2 form a continuum in the rate and degree of CPE produced in cell culture.^{48, 85, 101} At one extreme, foci of CPE may remain small (6 to 12 cells), non-progressive, and difficult to reproduce by passage of either the cell culture supernatant or infected cells. At the other extreme, some isolates of EHV-2 cause rapid and complete destruction of the cell monolayer after three to four days. Because the development of CPE by EHV-2/EHV-5 is, with most isolates, slow, passage of the virus may be necessary to achieve infection of the majority of cells in a monolayer. This is most successfully done through dispersal of infected cells from each focus of infection within the monolayer, by gently lifting the cells with trypsin-EDTA, followed by their transfer into a new culture flask containing fresh medium.^{75, 101} Co-culture of blood leukocytes appears to be more sensitive after four passages than direct PCR from buffy coat samples.¹¹⁷ Definitive identification of EHV-2 and EHV-5 isolated in cell culture can be made by reactivity with specific reference antiserum, PCR, or restriction endonuclease analysis of the viral DNA. PCR from cell lysates may provide confirmation even in the absence of visible CPE.¹³ Because rabbit antiserum against EHV-2 can be strain-specific when used in the virus neutralization test, immunofluorescence provides a more reliable method for seroidentification of equine gammaherpesvirus isolates.^{48, 69, 77}

Methods for direct detection of EHV-2 and EHV-5-specific DNA in clinical specimens (nasal secretions; peripheral blood leukocytes) by PCR and, most recently, real-time quantitative PCR (qPCR) have been reported.^{4, 7, 16, 34, 38, 43, 60, 84, 86, 96, 125} Because designed PCR primers may be specific for subpopulations of gammaherpesviruses, it is believed that virus detection by PCR underestimates the true frequency of infection.⁴³

The close antigenic relationship between EHV-2 and EHV-5 makes serological testing difficult. While complement fixation and virus neutralization tests have been described, an ELISA that can differentiate both viruses has been  developed.¹⁰⁹ The examination of paired acute and convalescent sera of young foals by virus neutralization test or ELISA , for specific seroconversion has, in some instances been useful in confirming recent infection by EHV-2.^{14, 44, 69, 70, 75, 110} As virtually all foals acquire EHV-2 infection by six to eight months of age and remain persistently or latently infected for long periods, serology is of limited value in establishing a diagnosis of active equid gammaherpesvirus reinfection beyond the time of acquisition of the primary infection. Furthermore, post-infection neutralizing antibody to EHV-2 is frequently of low titre, transitory in nature, and may fail to neutralize the specific heterologous reference strain used in the virus neutralization test.^{68, 79} In most cases, virus isolation is more effective than serological tests for detecting infections by slowly growing herpesviruses.^{69, 91, 110}

Figure 5 (A-D) Cytopathic effect induced by EHV-2 in low passage rabbit kidney 13 (RK-13) cells between 2- and 5-days post-inoculation. CPE is visible 3 days post-inoculation with EHV-2 strain T939. There is formation of syncytia and evidence of cell lysis.

Control

As for other herpesviruses, establishment of latency and periodic reactivation is a major challenge for infection control. In addition, exposure to both EHV-2 and EHV-5 occur very early in the horse’s life and despite the presence of maternal antibodies. While reactivation and shedding of equid alphaherpesviruses can be triggered by stress-related events and/or administration of corticosteroids, the mechanisms governing reactivation of gammaherpesviruses remain poorly understood but expected to be triggered by similar factors as those inducing reactivation of alphaherpesviruses. One factor that has been examined was shedding following transportation, and both EHV-2 and EHV-5 were the most commonly detected equine herpesviruses in nasal swabs following transportation.^{71, 93} This suggests that increased shedding of gammaherpesviruses is likely associated with stress-associated reactivation, and consequently generates conditions supportive of horizontal transmission. Thus, limiting stressful conditions is important to reduce the possibility of periodic shedding and further transmission. Based on the ubiquitous detection of EHV-2 and EHV-5 even in clinically healthy horses,  quarantine following a positive detection is not a recommended or needed measure.³⁹ However, biosecurity measures in recently transported animals are recommended, and a risk assessment of the population in question should be performed for decision-making.

On the basis of evidence suggesting that EHV-2 infection can play an aetiological role in predisposing foals to subsequent R. equi pneumonia, both passive immunization with hyperimmune equine serum against EHV-2 and active immunization with an ISCOM vaccine containing EHV-2 glycoprotein antigens have been used, with reported success, for the prophylactic treatment of annual recurrence of this highly fatal foal disease.^{11, 74}

Ocular disease in foals associated with infection by EHV-2 on breeding farms has been successfully treated with ophthalmic ointments containing either idoxuridine or trifluridine together with antibiotics and non-steroidal anti-inflammatory agents.^{29, 57, 63, 66} Treatment of herpetic conjunctivitis with glucocorticosteroids is controversial because of its potential suppressive effect on ocular immunity. In one study, the use of topical corticosteroid therapy for foal keratoconjunctivitis resulted in its recrudescence and failure to heal.¹⁰⁶

Specific therapeutic or prophylactic options for other EHV-2 infections generally have not been explored. Therapy for such infections is primarily supportive, with treatment decisions guided by the specific clinical presentation of individual cases.

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