Epidemiology of equine herpesviruses (EHVs) in Ethiopian equids and invasion characteristics of EHV-1 and EHV-3 in respiratory and genital mucosae

The equine herpesviruses (EHVs) are ubiquitous pathogens in equine populations worldwide. They establish a lifelong latent infection, which ensures the survival of herpesviruses in equine populations. The currently available modified live and inactivated products of EHV-1 and EHV-4 vaccines are not reliably providing protection against infection. A better understanding of the epidemiology of EHV infections in natural outbreaks is particularly important to formulate an effective intervention strategy. However, many data gaps exist and more investigation needs to be done. It is very well known that the respiratory mucosal epithelium is the primary site of EHV-1 replication. However, EHV-1 infection in the vaginal mucosa is largely unknown. Moreover, information regarding the pathogenesis of EHV-3 in the nasal and vaginal mucosa is limited

Replication characteristics of equine herpesvirus 1 and equine herpesvirus 3: comparative analysis using ex vivo tissue cultures

Replication kinetics and invasion characteristics of equine herpesvirus-1 and -3 (EHV-1/-3) in nasal and vaginal mucosae were compared using explants. The explants were cultured during 96 h with little change in viability. The tissues were inoculated with EHV-1 03P37 (neuropathogenic), 97P70 (abortigenic) and EHV-3 04P57, collected at 0, 24, 48 and 72 h post inoculation (pi) and stained for viral antigens. Both EHV-1 and EHV-3 replicated in a plaquewise manner. The plaques were already observed at 24 h pi, their size increased over time and did not directly cross the basement membrane (BM). However, EHV-1 infected the monocytic cells (MC) and hijacked these cells to invade the lamina propria. In contrast, EHV-3 replication was fully restricted to epithelial cells; the virus did not breach the BM via a direct cell-to-cell spread nor used infected MC. EHV-1-induced plaques were larger in nasal mucosa compared to vaginal mucosa. The opposite was found for EHV-3-induced plaques. Both EHV-1 strains replicated with comparable kinetics in nasal mucosa. However, the extent of replication of the abortigenic strain in vaginal mucosa was significantly higher than that of the neuropathogenic strain. Two-to-five-fold lower numbers of EHV-1-infected MC underneath the BM were found in vaginal mucosa than in nasal mucosa. Our study has shown that (i) EHV-1 has developed in evolution a predisposition for respiratory mucosa and EHV-3 for vaginal mucosa, (ii) abortigenic EHV-1 replicates better in vaginal mucosa than neuropathogenic EHV-1 and (iii) EHV-3 demonstrated a strict epithelial tropism whereas EHV-1 in addition hijacked MC to invade the lamina propria

Equine herpesvirus-1 myeloencephalopathy, an emerging threat of working equids in Ethiopia

Although equine herpesvirus myeloencephalopathy (EHM) is a sporadic and relatively uncommon manifestation of equine herpesvirus-1 (EHV-1), it has the potential for causing devastating outbreaks in horses. Up till now, there were no reported EHM outbreaks in donkeys and mules. This study describes the isolation and molecular characterization of EHV-1 from clinically EHM-affected horses (n = 6), mules (n = 3) and donkeys (n = 82) in Ethiopia during outbreaks from May 2011 to December 2013. The incidence of EHM cases was higher from April to mid-June. EHM in donkeys was more severe and death without clinical signs of paralysis, and recumbency was frequently observed. The main age of affected equines ranged from 7 to 10 years (n = 51; 56.0%), and females (n = 58; 63.7%) were more affected than males. The incidence of neuropathogenic (D752) and non-neuropathogenic (N752) variants of EHV-1 from EHM-affected equines in Ethiopia was assessed by sequencing the DNA polymerase gene (ORF30) of the EHV-1 isolates. The results indicated that from the total of 91 clinically affected equines, 90 (98.9%) of them had an ORF30 D752 genotype. An ORF30 N752 variant was only found in one donkey. Analysis of ORF68 as grouping marker for geographical differences showed that the Ethiopian EHV-1 isolates belong to geographical group 4. Due to the fatal nature of EHV-1 in donkeys, it would be interesting to examine the pathogenesis of EHM in this species. At present, there is no vaccine available in Ethiopia, and therefore, outbreaks of EHV-1 should be controlled by proper management adaptations. In addition, it is important to test the efficacy of the commercial vaccines not only in horses, but also in donkeys and mules

Dual infections of equine herpesvirus 1 and equine arteritis virus in equine respiratory mucosa explants

Equine herpesvirus 1 (EHV-1) and equine arteritis virus (EAV) induce respiratory problems and abortion in horses and are considered as two serious threats to equine industry. Both EHV-1 and EAV misuse patrolling leukocytes in the upper respiratory tract to breach the basement membrane (BM) and to migrate to blood vessels. So far, the behavior and impact of a double infection in the respiratory mucosa of a horse are unknown. In the present study, the outcome of double infections with EHV-1 and the low virulent EAV strain 08P187 (superinfection with an interval of 12 h or co-infection) were compared with single infections in fully susceptible RK-13 cells and equine upper respiratory mucosa explants. When RK-13 cells were inoculated with either EHV-1 or EAV 12 h prior to the subsequent EAV or EHV-1 inoculation, the latter EAV or EHV-1 infection was clearly suppressed at 24 hpi or 36 hpi, respectively, without EHV-1 and EAV co-infecting the same RK-13 cells. After simultaneous infection with EHV-1 and EAV, higher numbers of EAV infected cells but similar numbers of EHV-1 infected cells were found compared to the single infections, with a low number of EHV-1 and EAV co-infected RK-13 cells at 48 hpi and 72 hpi. In the upper respiratory mucosa exposed to EAV 12 h prior to EHV-1, the number and size of the EHV-1-induced plaques were similar to those of the EHV-1 single infected mucosa explants. In nasal and nasopharyngeal mucosae, EAV and EHV-1 pre-infections slightly reduced the number of EHV-1 and EAV infected leukocytes compared to the single infections and co-infection. In double EAV and EHV-1 infected explants, no co-infected leukocytes were detected. From these results, it can be concluded that EAV and EHV-1 are only slightly influencing each other's infection and that they do not infect the same mucosal leukocytes

Detection of equine herpesvirus (EHV) -1, -2, -4 and -5 in Ethiopian equids with and without respiratory problems and genetic characterization of EHV-2 and EHV-5 strains

Infections with equine herpesviruses (EHVs) are widespread in equine popula- tions worldwide. Whereas both EHV-1 and EHV-4 produce well-documented respiratory syndromes in equids, the contribution of EHV-2 and EHV-5 to disease of the respiratory tract is still enigmatic. This study describes the detection and genetic characterization of EHVs from equids with and without clinical respiratory disease. Virus-specific PCRs were used to detect EHV-1, -2, -4 and -5. From the total of 160 equids with respiratory disease, EHV-5 was detected at the highest prevalence (23.1%), followed by EHV-2 (20.0%), EHV-4 (8.1%) and EHV-1 (7.5%). Concurrent infections with EHV-2 and EHV-5 were recorded from nine (5.2%) diseased horses. Of the total of 111 clinically healthy equids, EHV-1 and EHV-4 were never detected whereas EHV-2 and EHV-5 were found in 8 (7.2%) and 18 (16.2%) horses, respec- tively. A significantly higher proportion of EHV-2-infected equids was observed in the respiratory disease group (32/160, 20.0%; P = 0.005) com- pared to those without disease (8/111; 7.2%). EHV-2-positive equids were three times more likely to display clinical signs of respiratory disease than EHV-2-negative equids (OR 3.22, 95% CI: 1.42–7.28). For EHV-5, the observed difference was not statistically significant (P = 0.166). The phyloge- netic analysis of the gB gene revealed that the Ethiopian EHV-2 and EHV-5 strains had a remarkable genetic diversity, with a nucleotide sequence identity among each other that ranged from 94.0 to 99.4% and 95.1 to 100%, respec- tively. Moreover, the nucleotide sequence identity of EHV-2 and EHV-5 with isolates from other countries acquired from GenBank ranged from 92.9 to 99.1% and 95.1 to 99.5%, respectively. Our results suggest that besides EHV- 1 and EHV-4, EHV-2 is likely to be an important contributor either to induce or predispose equids to respiratory disease. However, more work is needed to better understand the contribution of EHV-2 in the establishment of respiratory disease