Detection of equine herpesvirus antibodies in large-scale donkey farms in Liaocheng area.

BACKGROUND: Equine herpesvirus (EHV) can cause respiratory, reproductive and neurological diseases in equine animals, including donkeys. The main pathogens responsible for these diseases are EHV type 1 (EHV-1) and EHV-4. In this study, we collected serum samples from 230 donkeys on 27 large-scale donkey farms to detect EHV-1 and EHV-4 antibodies. We analyzed the presence of EHV antibodies based on region, age and season. RESULTS: Out of the 27 farms, 62.96% (17/27) tested positive for EHV. Of the 230 donkeys tested, 2.61% (6/230) were positive only for EHV-1, 5.22% (12/230) were positive only for EHV-4, and 4.78% (11/230) were positive for both EHV-1 and EHV-4. The highest percentage of positive donkeys (21.28%) was found in Dong'e County. The seropositivity rate among donkeys aged 1-4 years was significantly higher compared to the group of donkeys aged 0-1 year (p < 0.05). Additionally, the positive rate was significantly higher in fall and winter compared to spring and summer (p < 0.05). CONCLUSIONS: Altogether, our findings indicate that large-scale donkey farms in the Liaocheng area have a high prevalence of EHV antibodies. Since Liaocheng is an important donkey trading market in Shandong Province, it is crucial to consider the risk of disease transmission based on our test results. This will help in early detection and prevention of EHV outbreaks.

Outbreak of equine herpesvirus 4 (EHV-4) in Denmark: tracing patient zero and viral characterization.

BACKGROUND: Equine herpesvirus 4 (EHV-4) causes respiratory disease in horses, and the virus is considered endemic in the global equine population. However, outbreaks can occur when several horses are gathered in relation to shows, competitions, breeding units and at hospitals. In the spring year 2022, an EHV-4 outbreak occurred at the Large Animal Teaching Hospital, University of Copenhagen, Denmark. Nine horses were tested EHV-4 positive during the outbreak, which lasted approx. seven weeks. In addition, a tenth horse "Eq10" tested EHV-4 positive almost three weeks after the last of the outbreak horses tested positive. Detailed clinical registrations were obtained from all ten horses as well as their location and movement during hospitalization. Nasal swabs were obtained throughout the outbreak and tested by qPCR for EHV-4. Additionally, pre- and post-infection sera were tested for the presence of EHV-4 antibodies. Selected samples were characterized by partial and full genome sequencing. RESULTS: The most common clinical signs of the EHV-4 infected horses during this outbreak were pyrexia, nasal discharge, mandibular lymphadenopathy and increased lung sounds upon auscultation. Based on the locations of the horses, EHV-4 detection and antibody responses the most likely "patient zero" was identified as being "Eq1". Partial genome sequencing revealed that Eq10 was infected by another wild type EHV-4 strain, suggesting that the hospital was able to eliminate the outbreak by testing and reinforcing biosecurity measures. The complete genome sequence of the outbreak strain was obtained and revealed a closer relation to Australian and Japanese EHV-4 strains rather than to other European EHV-4 strains, however, very limited sequence data are available from Europe. CONCLUSION: The study illustrated the transmission of EHV-4 within an equine facility/hospital and provided new insights into the viral shedding, antibody responses and clinical signs related to EHV-4 infections. Finally, sequencing proved a useful tool in understanding the transmission within the hospital, and in characterizing of the outbreak strain.

Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1.

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored.

A Screening Study Identified Decitabine as an Inhibitor of Equid Herpesvirus 4 That Enhances the Innate Antiviral Response.

Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.

Effect of dexamethasone on antibody response of horses to vaccination with a combined equine influenza virus and equine herpesvirus-1 vaccine.

BACKGROUND: Dexamethasone is routinely administered to horses but its effect on the antibody response to a commercial EIV/EHV vaccine is unclear. HYPOTHESIS: Horses receiving dexamethasone will have lower postvaccination antibody levels against EIV and EHV-1 than vaccinated controls. ANIMALS: Fifty-five healthy adult research horses. METHODS: Randomized cohort study. Control (no vaccine, group 1), vaccination only (EIV/EHV-1/EHV-4, Prestige 2, Merck Animal Health, group 2), vaccination and concurrent single intravenous dose of dexamethasone (approximately .05 mg/kg, group 3), vaccination and 3 intravenous doses of dexamethasone at 24 hours intervals (group 4). Serum SAA levels were measured on day 1 and day 3. Antibody levels against EIV (hemagglutination inhibition assay, Kentucky 2014 antigen) and EHV-1 (multiplex ELISA targeting total IgG and IgG 4/7) were measured on day 1 and day 30. RESULTS: Significantly increased mean antibody titers after vaccination were only noted against EIV and only after the vaccination alone (n = 14, prevaccine mean [prvm] 166.9, SD 259.6, 95% CI 16.95-316.8; postvaccine mean [povm] 249.1, SD 257.2, 95% confidence interval [CI] 100.6-397.6, P = .02) and the single dose dexamethasone (n = 14, prvm 93.14, SD 72.2, CI 51.45-134.8; povm 185.1, SD 118, CI 116.7-253.6, P = .01), but not after multiple doses of dexamethasone (n = 14, prvm 194.3, SD 258.3, CI 45.16-343.4; povm 240.0, SD 235.7, CI 103.9-376.1, P > .05). CONCLUSION: The effect of dexamethasone on the postvaccine antibody response varies depending on the dosing frequency and the antigen-specific antibody type.

Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay.

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 °C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests.

Multiplex real-time PCR for the detection and differentiation of equid gammaherpesvirus 2 and 5.

Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Analytical sensitivity was assessed by standard curve generation of serial plasmid dilutions containing the respective target gene. Melting curves and BLAST analysis of the sequences indicated specific detection of the viruses. The lower limit of detection of the singleplex rtPCR was 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively. Comparison of the Ct values of a selection of positive field samples showed only minimal differences between the singleplex and the multiplex assay. The here described multiplex rtPCR protocol allows sensitive and specific detection of EHV-2 and EHV-5. It represents a convenient and rapid tool for future studies to investigate the clinical relevance of EHV-2 and EHV-5 in more detail.

Frequency of Detection of Respiratory Pathogens in Nasal Secretions From Healthy Sport Horses Attending a Spring Show in California.

The objective of this study was to determine detection frequency of respiratory viruses (equine influenza virus [EIV], equine herpesvirus-1 [EHV-1], EHV-2, EHV-4, EHV-5, equine rhinitis A virus [ERAV], ERBV) and bacteria (Streptococcus equi ss. equi[S. equi], S. equi ss. zooepidemicus[S. zooepidemicus]) in 162 nasal secretions and 149 stall swabs from healthy sport horses attending a spring show in California. Nasal and stall swabs were collected at a single time point and analyzed using qPCR. The detection frequency of respiratory pathogens in nasal secretions was 38.9% for EHV-2, 36.4% for EHV-5, 19.7% for S. zooepidemicus, 1.2% for ERBV, 0.6% for S. equi and 0% for EIV, EHV-1, EHV-4 and ERAV. The detection frequency of respiratory pathogens in stall swabs was 65.8% for S. zooepidemicus, 33.5% for EHV-2, 27.5% for EHV-5, 3.3% for EHV-1, 1.3% for EHV-4 and 0% for EIV, ERAV, ERBV and S. equi. Commensal viruses and bacteria were frequently detected in nasal secretions and stall swabs from healthy sport horses. This was in sharp contrast to the subclinical shedding of well-characterized respiratory pathogens. Of interest was the clustering of five EHV-1 qPCR-positive stalls from apparently healthy horses with no evidence of clinical spread. The results highlight the role of subclinical shedders in introducing respiratory pathogens to shows and their role in environmental contamination. The results also highlight the need to improve cleanliness and disinfection of stalls utilized by performance horses during show events.

Immune and Inflammatory Response in Horse Vaccinated Against Equine Herpesviruses 1 (EHV-1) and 4 (EHV-4) Assessed by Serum Protein Electrophoretic Pattern and Leukocyte Population.

Protection against infectious diseases can be obtained with vaccines generating immunogenic response through a combination of humoral and cellular immunity. In this study haematological and serum protein electrophoretic profiles of horses vaccinated against herpesvirus 1 (EHV-1) and 4 (EHV-4) were evaluated. Blood samples were collected from 16 horses before (T0), after 24h, 48h, 72h, 1st week, 2nd week and 3rd week (T1I, T2I, T3I, T7I, T14I and T21I) from the first EHV vaccine-dose administration as well as before (TPREII), and after 24h, 48h, 72h, 1st week, 2nd week, 3rd week and 4th week (T1II, T2II, T3II, T7II, T14II, T21II and T28II) from the EHV vaccine-booster. Total leukocyte values increased at T1I, T1II, T3II and T28II compared to T0 (P < .01). Higher lymphocytes and lower neutrophils values were found after first vaccine-dose and vaccine-booster administration compared to the T0 (P < .01). Monocytes showed higher values at T14II than T0 (P < .01). Higher serum values of total proteins, α1-, α-2-, ß1-, ß2- and γ-globulins were found in horses after first vaccine-dose and vaccine-booster administration (P < .01). Gathered results suggest that horses vaccinated against EHV1 and EHV-4 exhibited a dynamic change of WBC, lymphocytes, neutrophils and monocytes. Moreover, the analysis of serum electrophoresis pattern suggested that EHV vaccination induced the development of inflammation and antibody response in vaccinated horses as highlighted by the increase of α-, ß- and γ-globulin fractions. These changes probably reflect the systemic immunological adaptation of animals to EHV vaccine.

Kinetics of the Equid Herpesvirus 2 and 5 Infections among Mares and Foals from Three Polish National Studs.

Equid herpesvirus 2 (EHV-2) and 5 (EHV-5) are two γ-herpesviruses that are commonly detected from horses worldwide, based on several cross-sectional molecular surveys. Comparatively few studies examined the dynamics of γ-herpesvirus infection over time in a group of horses. The aim of the current study was to investigate the dynamics of EHV-2/5 infections among mares and their foals at three Polish national studs with different breeds of horses: Arabians, Thoroughbreds and Polish Konik horses. Nasal swabs were collected from each of 38 mare-foal pairs monthly for a period of 6 to 8 months. Virus-specific quantitative PCR assays were used to determine the viral load of EHV-2 and EHV-5 in each sample. All 76 horses sampled were positive for EHV-2 or EHV-5 on at least one sampling occasion. The majority (73/76, 96%) were infected with both EHV-2 and EHV-5. In general, the mean load of viral DNA was higher in samples from foals than from mares, but similar for EHV-2 and EHV-5 at most sampling occasions. There was, however, a considerable variability in the viral DNA load between samples collected at different times from the same foal, as well as between samples from different foals. The latter was more apparent for EHV-2 than for EHV-5. All foals became infected with both viruses early in life, before weaning, and remained positive on all, or most, subsequent samplings. The virus shedding by mares was more intermittent, indicating the existence of age-related differences. Overall, the data presented extend our knowledge of EHV-2/5 epidemiology among mares and foals.

Outbreak of neuropathogenic equid herpesvirus 1 causing abortions in Yili horses of Zhaosu, North Xinjiang, China.

BACKGROUND: EHV-1 is one of the most serious viral pathogens that frequently cause abortion in horses around the world. However, so far, relatively little information is available on EHV-1 infections as they occur in China. In January 2021, during an abortion storm which occurred in Yili horses at the Chinese State Studs of Zhaosu (North Xinjiang, China), 43 out of 800 pregnant mares aborted. RESULTS: PCR detection revealed the presence of EHV-1 in all samples as the possible cause of all abortions, although EHV-4, EHV-2 and EHV-5 were also found to circulate in the aborted fetuses. Furthermore, the partial ORF33 sequences of the 43 EHV-1 shared 99.3-100% and 99.0-100% similarity in nucleotide and amino acid sequences respectively. These sequences not only indicated a highly conserved region but also allowed the strains to group into six clusters. In addition, based on the predicted ORF30 nucleotide sequence, it was found that all the strains carried a guanine at the 2254 nucleotide position (aspartic acid at position 752 of the viral DNA polymerase) and were, therefore, identified as neuropathogenic strains. CONCLUSION: This study is the first one that establishes EHV-1 as the cause of abortions in Yili horses, of China. Further characterization of the ORF30 sequences revealed that all the EHV-1 strains from the study carried the neuropathogenic genotype. Totally, neuropathogenic EHV-1 infection in China's horse population should be concerned although the virus only detected in Yili horse abortions.

The equine mesenchymal stromal cell secretome inhibits equid herpesvirus type 1 strain Ab4 in epithelial cells.

Equid herpesvirus 1 (EHV-1) outbreaks occur when virus spreads from infected horses to in-contact horses, primarily via nasal shedding. This study evaluated the efficacy of factors secreted by equine peripheral blood derived mesenchymal stromal cells (PB-MSCs), collectively named the secretome, to inhibit the growth of EHV-1 in (i) 2D epithelial cell cultures (RK-13) in vitro, (ii) 3D equine nasal explants in vitro and (iii) an EHV-1 infection mouse model in vivo. The PB-MSC secretome was found to inhibit EHV-1 in RK-13 cells as well as in the epithelium of equine nasal explants. Although the PB-MSC secretome did not decrease overall severity of EHV-1 infection in mice, as determined by weight loss and viral titers in lungs, histological analyses indicated local reduction of EHV-1 infection in nasal epithelium. These results indicate that the PB-MSC secretome inhibits EHV-1 in epithelial cells in a context-dependent manner.

Smartphone-based multiplex 30-minute nucleic acid test of live virus from nasal swab extract.

Rapid, sensitive and specific detection and reporting of infectious pathogens is important for patient management and epidemic surveillance. We demonstrated a point-of-care system integrated with a smartphone for detecting live virus from nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in ∼30 minutes.

The effect of equine herpesvirus type 4 on type-I interferon signaling molecules.

Equine herpesvirus type 4 (EHV-4) is mildly pathogenic but is a common cause of respiratory disease in horses worldwide. We previously demonstrated that unlike EHV-1, EHV-4 is not a potent inducer of type-I IFN and does not suppress that IFN response, especially during late infection, when compared to EHV-1 infection in equine endothelial cells (EECs). Here, we investigated the impact of EHV-4 infection in EECs on type-I IFN signaling molecules at 3, 6, and 12 hpi. Findings from our study revealed that EHV-4 did not induce nor suppress TLR3 and TLR4 expression in EECs at all the studied time points. EHV-4 was able to induce variable amounts of IRF7 and IRF9 in EECs with no evidence of suppressive effect on these important transcription factors of IFN-α/ß induction. Intriguingly, EHV-4 did interfere with the phosphorylation of STAT1/STAT2 at 3 hpi and 6 hpi, less so at 12 hpi. An active EHV-4 viral gene expression was required for the suppressive effect of EHV-4 on STAT1/STAT2 phosphorylation during early infection. One or more early viral genes of EHV-4 are involved in the suppression of STAT1/STAT2 phosphorylation observed during early time points in EHV-4-infected EECs. The inability of EHV-4 to significantly down-regulate key molecules of type-I IFN signaling may be related to the lower severity of pathogenesis when compared with EHV-1. Harnessing this knowledge may prove useful in controlling future outbreaks of the disease.

Epizootiological investigation of equine herpesvirus type 1 infection among Japanese racehorses before and after the replacement of an inactivated vaccine with a modified live vaccine.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) infection is a major cause of pyrexias in winter among Japanese racehorses. In 2014-2015, the Japan Racing Association (JRA) changed the EHV-1 vaccine from an inactivated vaccine to a live vaccine (both produced by Nisseiken). To evaluate the effect of changing the vaccines, the capacities of these vaccines to induce virus-neutralizing (VN) antibodies were compared, and an epizootiological investigation of EHV-1 was performed at the JRA Ritto Training Center during epizootic periods from 2010-2011 to 2016-2017. RESULTS: Three-year-old horses that received the first dose of live vaccine showed higher geometric mean (GM) VN titers (205 and 220) than those that received inactivated vaccine (83, P < 0.05). The response rates after vaccination with the live vaccine (76 and 90%) were higher than that after vaccination with inactivated vaccine (42%, P < 0.05). Four-year-old horses from 2015 to 2017 that had received the live vaccine in the previous epizootic periods had higher GM titers (205 to 246) than those from 2011 to 2014, which had received the inactivated vaccine (139 to 164, P < 0.05). The estimated numbers of horses infected with EHV-1 or EHV-4, or both, in 2011-2012 (29 [95%CI: 21-37]) and 2013-2014 (37 [95%CI: 27-47]) were higher than those in the other periods (7 [95%CI: 2-12] to 16 [95%CI: 9-23]). Likewise, the seroconversion rates to EHV-1 in horses that stayed at the training center in 2011-2012 (66.0%) and 2013-2014 (52.0%) were higher than those in the other periods (12.0 to 28.6%). CONCLUSIONS: The live EHV-1 vaccine is highly immunogenic and provides greater VN antibody responses than the inactivated vaccine. Unlike the period when the policy was to use inactivated vaccine, there was no detectable epizootic EHV-1 infection at the training center during three consecutive periods after the introduction of the live vaccine. These results suggest that the replacement of inactivated vaccine with live vaccine, together with the achievement of high vaccination coverage, reinforced the herd effect, and contributed to better control of EHV-1 epizootics in the training center.

Failure to detect equid herpesvirus types 1 and 4 DNA in placentae and healthy new-born Thoroughbred foals.

Equid herpesvirus type 1 is primarily a respiratory tract virus associated with poor athletic performance that can also cause late gestation abortion, neonatal foal death and encephalomyelopathy. Horizontal transmission is well described, whereas evidence of vertical transmission of equid herpesvirus type 1 associated with the birth of a healthy foal has not been demonstrated. This study sampled a population of Thoroughbred mares (n = 71), and their healthy neonatal foals and foetal membranes, to test for the presence of both equid herpesvirus types 1 and 4 using a quantitative polymerase chain reaction assay. Foetal membrane swabs and tissue samples were taken immediately post-partum, and venous blood samples and nasal swabs were obtained from both mare and foal 8 h after birth. Neither equid herpesvirus type 1 nor equid herpesvirus type 4 nucleic acid was detected in any sample, and it was concluded that there was no active shedding of equid herpesvirus types 1 and 4 at the time of sampling. Consequently, no evidence of vertical transmission of these viruses could be found on this stud farm during the sampling period.

Screening and evaluation of antiviral compounds against Equid alpha-herpesviruses using an impedance-based cellular assay.

Equid alpha-herpesviruses (EHV) are responsible for different diseases in equine population. EHV-1 causes respiratory diseases, abortions and nervous disorders, EHV-4 causes respiratory diseases and sporadic abortion, while EHV-3 is responsible of equine coital exanthema. In view of the lack of efficacy of vaccines against EHV-1 and EHV-4 and in the absence of vaccines against EHV-3, the use of antiviral treatment is of great interest. In this study, we documented the interest of the Real-Time Cell Analysis (RTCA) technology to monitor the cytopathic effects induced by these viruses on equine dermal cells, and established the efficacy of this method to evaluate the antiviral effect of aciclovir (ACV) and ganciclovir (GCV). In addition, the RTCA technology has also been found appropriate for the high-throughput screening of small molecules against EHV, allowing the identification of spironolactone as a novel antiviral against EHV.

Detection of equine herpesvirus 1 and 4 and its association with latency-associated transcripts in naturally infected horses in Colombia / Detección de los herpesvirus equinos 1 y 4 y su relación con transcriptos asociados a la latencia en caballos infectados naturalmente en Colombia

ABSTRACT Objective. Determine the presence of antibodies and viral genomes of EHV-1 and EHV-4, as well as to detect the presence of latency associated transcripts (LATs) in a selected population of Colombian horses. Materials and methods. Serum samples, submandibular lymph nodes and trigeminal ganglion were obtained from 50 horses and analyzed. Sera were evaluated for the presence of antibodies against EHV-1 and EHV-4 while tissues were initially evaluated for the presence of viral genome by nPCR. Finally, samples were used for the detection of LATs through RT-PCR. Results. In general, 6/50 samples showed antibodies to EHV-1 and 44/50 were positive for EHV-4. As for viral genome detection, 10/50 samples were positive for EHV-1 and 30/50 were positive for EHV-4; in addition, 22/35 horses positive for EHV DNA were positive for LATs. The use of these tests led to eight possible combinations of results. Conclusions. The evidence used shows that horses can have simple viral infection, co-infections with both viruses, latency due to the presence of LATs and the simultaneous presence of LATs and viral genome replication at a given time. It contributes to the understanding of the behavior of the disease in Colombia and calls attention to the importance of implementing complementary diagnoses to the serology for the control of these viruses.

RESUMEN Objetivo. Determinar presencia de anticuerpos y genoma viral de EHV-1 y EHV-4, como también detectar la presencia de transcriptos asociados a latencia (LATs) en una población seleccionada de caballos colombianos. Materiales y métodos. Muestras de suero, nódulos linfáticos submandibulares y ganglio trigémino se obtuvieron de 50 caballos y fueron analizadas. Los sueros se evaluaron para la presencia de anticuerpos contra EHV-1 y EHV-4 mientras que los tejidos se evaluaron inicialmente para la presencia de genoma viral por nPCR. Finalmente, las muestras se emplearon para la detección de LATs a través de RT-PCR. Resultados. En general, 6/50 muestras mostraron anticuerpos para EHV-1 y 44/50 fueron positivos para EHV-4. En cuanto a la detección del genoma viral, 10/50 muestras fueron positivas para EHV-1 y 30/50 fueron positivas para EHV-4; además, 22/35 caballos positivos para DNA de EHV fueron positivos para LATs. El empleo de estas pruebas llevó a ocho posibles combinaciones de resultados. Conclusiones. Se confirma la presencia de estos virus en la población equina colombiana. Las pruebas empleadas demuestran que los caballos pueden tener infección viral simple, co-infecciones con ambos virus, estado de latencia debido a la presencia de los LATs y presencia simultánea de LATs y de replicación de genoma viral en un momento dado. Se aporta al entendimiento del comportamiento de la enfermedad en Colombia y se llama la atención sobre la importancia de implementar diagnósticos complementarios a la serología para el control de estos virus.

Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses.

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R2 values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.