Virological and molecular biological investigations into equine herpes virus type 2 (EHV-2) experimental infections.

Two 18-month-old naturally reared ponies were used to investigate the pathogenicity of EHV-2. After dexamethasone treatment, pony 1 was inoculated intranasally with EHV-2 strain T16, which has been isolated from a foal with keratoconjunctivitis superficialis and pony 2 was similarly inoculated with strain LK4 which was originally isolated from a horse with upper respiratory tract disease. Following virus inoculation, pyrexia was not detected in either pony but both developed conjunctivitis, lymphadenopathy, and coughing. EHV-2 was detected in nasal mucus samples up to day 12 post infection (p.i.), in eye swabs up to day 10 p.i., and in buffy coat cells throughout the investigation in both animals. EHV-2-specific antibody titres were raised significantly 18 days p.i. Following the administration of dexamethasone, 3 months p.i., infectious virus was again detected in nasal mucus and conjunctival swabs from both ponies for 7 days. The tissue distribution of EHV-2 genome was studied post mortem, by means of a nested PCR. EHV-2 was detected in lymphoid tissues, lung, conjunctiva, trigeminal ganglia and olfactory lobes of pony 2, whereas in pony 1 only the conjunctiva of the left eye was PCR positive.

Equine herpesvirus 4 DNA in trigeminal ganglia of naturally infected horses detected by direct in situ PCR.

Neuronal and lymphoid tissues of 15 randomly selected horses were analysed post mortem by liquid nested-PCR to study the tropism of equine herpesvirus 4 (EHV-4). In four animals the trigeminal ganglia and in one case the lung were positive. Using a direct in situ PCR the EHV-4 genome was localized in the nuclei of neurons and in the bronchiolar as well as alveolar epithelium of the lung. In none of these tissues could infectious virus or viral antigens be detected. Applying the more sensitive liquid RT-PCR, however, an acute infection was demonstrated in one of the trigeminal ganglia by amplification of viral transcripts coding for glycoprotein B. The failure to detect these transcripts in the other trigeminal ganglia and the lung indicates a latent infection. This report formally proves that, like other members of the Alpha-herpesvirinae, EHV-4 establishes latency in the trigeminal ganglia.

Detection and distribution of equine herpesvirus 2 DNA in the central and peripheral nervous systems of ponies.

The distribution of equine herpesvirus 2 (EHV-2) DNA within neurological and lymphoid tissues from 12 EHV-2 seropositive Welsh mountain ponies was determined by PCR. The lymphoid sites sampled in this study were almost universally PCR positive, thus confirming the existing virus co-cultivation data which suggest that the lymph nodes draining the respiratory tract are the main reservoirs of EHV-2 DNA. In addition, EHV-2 DNA was also detected, albeit with lower frequency, within both the peripheral and central nervous systems (PNS and CNS) of these animals. Of the CNS sites sampled 11% were PCR-positive and in the PNS the trigeminal ganglion proved PCR-positive in 50% of the animals tested. Since the nasal epithelium is innervated by the maxillary division of the trigeminal nerve, these observations suggest that the trigeminal ganglion may represent a biologically important site for EHV-2 latency.

Distribution and relevance of equine herpesvirus type 2 (EHV-2) infections.

Equine herpesvirus type 2 (EHV-2) is a slow-growing, cytopathogenic gammaherpesvirus, which is suggested to be ubiquitous in the equine population. However, its precise role as a pathogen and its tissue tropism remains uncertain. To estimate the prevalence of EHV-2 in Germany and to investigate the possible pathogenicity of the virus, peripheral blood leucocytes (PBL) from 172 horses were examined for EHV-2 DNA by a sensitive and specific nested PCR based on the EcoRI-N genomic fragment and by classical cocultivation. PBL samples from 51% of the horses were positive by PCR and virus was isolated from 31% of the horses by cocultivation. However, almost all animals were seropositive for EHV-2. This may indicate that PBL do not harbour EHV-2 indefinitely after infection. Furthermore, a correlation between clinical signs and EHV-2 as a causative agent could not be determined. Nevertheless, the prevalence of virus was high among horses with upper respiratory tract disease, abortion and severe ataxia. The products of the second round of the PCR reactions showed size polymorphism. Sequencing of the products revealed that these size differences were due to repetition of the motif (AGACAGGGGCCATGCTGGC) between 9-16 times depending on the isolate, suggesting that the nested PCR might be a useful tool for the differentiation of EHV-2 isolates.