Molecular testing for equine herpesviruses 1 (EHV-1) in healthy postpartum broodmares.

Objective: Our objective was to determine whether equine herpesviruses 1 (EHV-1) viral nucleic acids could be detected immediately after foaling from nasal and vaginal swabs, whole blood, and placental tissue of healthy mares. Animals procedure and results: Nasal and vaginal swabs, EDTA blood, and placental tissue (296 samples) were collected from 74 clinically healthy postpartum broodmares within 24 h after giving birth to live, clinically healthy foals. All samples were tested (PCR) for nucleic acids of neuropathogenic and non-neuropathogenic strains of EHV-1, and all were negative. Conclusion and clinical relevance: As EHV-1 was not detected in the immediate postpartum period in healthy mares with uncomplicated foaling, we inferred that EHV-1-positive samples from aborting mares and/or EHV-1 detection in fetal membranes indicate EHV-1-associated abortion.

Tests moléculaires pour l'herpèsvirus équin 1 (EHV-1) chez des juments poulinières post-partum en bonne santé. Objectif: Notre objectif était de déterminer si les acides nucléiques viraux de l'herpèsvirus équin 1 (EHV-1) pouvaient être détectés immédiatement après la mise bas à partir de prélèvements nasaux et vaginaux, de sang total et de tissus placentaires de juments saines. Animaux procédure et résultats: Des écouvillons nasaux et vaginaux, du sang EDTA et du tissu placentaire (296 échantillons) ont été prélevés sur 74 juments poulinières post-partum cliniquement saines dans les 24 heures suivant la naissance de poulains vivants et cliniquement sains. Tous les échantillons ont été testés (PCR) pour les acides nucléiques des souches neuropathogènes et non-neuropathogènes de l'EHV-1, et tous se sont révélés négatifs. Conclusion et pertinence clinique: Comme l'EHV-1 n'a pas été détecté dans la période post-partum immédiate chez des juments en bonne santé avec un poulinage sans complication, nous avons déduit que les échantillons positifs pour l'EHV-1 provenant de juments qui ont avorté et/ou la détection de l'EHV-1 dans les membranes foetales indiquent un avortement associé à l'EHV-1.(Traduit par Dr Serge Messier).

Investigation of the Use of Environmental Samples for the Detection of EHV-1 in the Stalls of Subclinical Shedders.

In populations of healthy show horses, the subclinical transmission and circulation of respiratory pathogens can lead to disease outbreaks. Due to recent outbreaks of equine herpesvirus-1 myeloencephalopathy (EHM) in the USA and Europe, many show organizers have instituted various biosecurity protocols such as individual horse testing, monitoring for early clinical disease and increasing hygiene and cleanliness protocols. The aim of this study was to determine the accuracy of detecting EHV-1 in the various environmental samples collected from the stalls of subclinical shedders. Four healthy adult horses were vaccinated intranasally with a modified-live EHV-1 vaccine in order to mimic subclinical shedding. Three additional horses served as non-vaccinated controls. All the horses were stabled in the same barn in individual stalls. Each vaccinated horse had nose-to-nose contact with at least one other horse. Prior to the vaccine administration, and daily thereafter for 10 days, various samples were collected, including a 6" rayon-tipped nasal swab, an environmental sponge, a cloth strip placed above the automatic waterer and an air sample. The various samples were processed for nucleic acid purification and analyzed for the presence of EHV-1 via quantitative PCR (qPCR). EHV-1 in nasal secretions was only detected in the vaccinated horses for 1-2 days post-vaccine administration. The environmental sponges tested EHV-1 qPCR-positive for 2-5 days (median 3.5 days) in the vaccinated horses and 1 day for a single control horse. EHV-1 was detected by qPCR in stall strips from three out of four vaccinated horses and from two out of three controls for only one day. EHV-1 qPCR-positive air samples were only detected in three out of four vaccinated horses for one single day. For the vaccinated horses, a total of 25% of the nasal swabs, 35% of the environmental stall sponges, 7.5% of the strips and 7.5% of the air samples tested qPCR positive for EHV-1 during the 10 study days. When monitoring the subclinical EHV-1 shedders, the collection and testing of the environmental sponges were able to detect EHV-1 in the environment with greater frequency as compared to nasal swabs, stationary strips and air samples.

Evaluation of Non-Invasive Sampling Techniques for the Molecular Surveillance of Equid Herpesviruses in Yearling Horses.

BACKGROUND: Equid alphaherpesvirus 1 (EHV-1) is a highly contagious respiratory tract pathogen of horses, and infection may be followed by myeloencephalopathy or abortion. Surveillance and early detection have focused on PCR assays using less tolerated nasal swabs. Here, we assess non-invasive non-contact sampling techniques as surveillance tools in naturally equid gammaherpesvirus 2-shedding horses as surrogates for EHV-1. METHODS: Horses were individually housed for 10 h periods on 2 consecutive days. Sampling included nasal swabs, nostril wipes, environmental swabs, droplet-catching devices, and air sampling. The latter was completed via two strategies: a combined air sample collected while going from horse to horse and a collective air sample collected at a stationary central point for 6 h. Samples were screened through quantitative PCR and digital PCR. RESULTS: Nine horses on day 1 and 11 horses on day 2 were positive for EHV-1; overall, 90.9% of the nostril wipes, 81.8% of the environmental surfaces, and 90.9% of the droplet-catching devices were found to be positive. Quantitative analysis showed that the mean DNA copies detection per cm2 of nostril wipe sampled concentration (4.3 × 105 per day) was significantly (p < 0.05) comparable to that of nasal swabs (3.6 × 105 per day) followed by environmental swabs (4.3 × 105 per day) and droplet catchers (3.5 × 103 per day), respectively. Overall, 100% of the air samples collected were positive on both qPCR and dPCR. In individual air samples, a mean concentration of 1.0 × 104 copies of DNA were detected in per m3 air sampled per day, while in the collective air samples, the mean concentration was 1.1 × 103. CONCLUSIONS: Environmental samples look promising in replacing direct contact sampling. Environmental and air sampling could become efficient surveillance tools at equestrian events; however, it needs threshold calculations for minimum detection levels.

Divergent strains of EHV-1 in Swedish outbreaks during 2012 to 2021.

Equid alphaherpesvirus 1 (EHV-1) is a ubiquitous and significant viral pathogen in horses worldwide, causing a range of conditions, including fever, respiratory disease, abortion in pregnant mares and the severe neurological disease called equine herpes myeloencephalopathy (EHM). Despite that EHV-1 is a notifiable animal disease in Sweden, there is limited knowledge about the circulating strains. This study aimed to analyze the genetic diversity of EHV-1 strains in equine samples from different Swedish outbreaks by partial genome sequencing. Genotyping based on three selected open reading frames ORF11, ORF30, and ORF34 in the viral genome was conducted for 55 outbreaks of EHV-1 spanning from the years 2012 to 2021. The analysis revealed 14 different genovariants, with one prominent genovariant identified in 49% of the outbreaks. Additionally, the study identified seven mutations not previously described. Three new mutations were demonstrated in ORF11, all synonymous, and four new mutations in ORF34, two synonymous, and two non-synonymous. Notably, different EHV-1 genovariants were found in five out of six studied EHM outbreaks, but clonal spreading was shown within the outbreaks. Moreover, the study demonstrated that healthy (recovered) horses that returned from an EHM outbreak at an international meeting in Valencia, Spain (2021), were positive for the virus clone responsible for the severe disease outbreak despite several weeks of quarantine. These findings shed light on the genetic diversity and transmission dynamics of the virus and significantly contribute to better understanding of the epidemiology of EHV-1 in Sweden and globally.

First identification and isolation of equine herpesvirus type 1 in aborted fetal lung tissues of donkeys.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) is commonly associated with horse abortion. Currently, there are no reported cases of abortion resulting from EHV-1 infection in donkeys. RESULTS: This was the first survey-based study of Chinese donkeys. The presence of EHV-1 was identified by PCR. This survey was conducted in Chabuchar County, North Xinjiang, China, in 2020. A donkey EHV-1 strain (Chabuchar/2020) was successfully isolated in MDBK cells. Seventy-two of 100 donkey sera were able to neutralize the isolated EHV-1. Moreover, the ORF33 sequence of the donkey-origin EHV-1 Chabuchar/2020 strain showed high levels of similarity in both its nucleotide (99.7‒100%) and amino acid (99.5‒100%) sequences, with those of horse EHV-1 strains. EHV-1 Chabuchar/2020 showed significant consistency and was classified within cluster 1 of horse EHV-1 strains. Further, analysis of the expected ORF30 nucleotide sequence revealed that donkey EHV-1 strains contained guanine at position 2254, resulting in a change to aspartic acid at position 752 of the viral DNA polymerase. Therefore, these strains were classified as horse neuropathogenic strains. Lastly, a phylogenetic tree was constructed using the partial ORF68 nucleotide sequences, showing that the identified donkey EHV-1 strain and the EHV-1 strain found in aborted Yili horses in China comprised a novel independent VIII group. CONCLUSION: This study showed the first isolation and identification of EHV-1 as an etiological agent of abortions in donkeys. Further analysis of the ORF33, ORF30, and ORF68 sequences indicated that the donkey EHV-1 contained the neuropathogenic genotype of strains in the VIII group. It is thus important to be aware of EHV-1 infection in the donkey population, even though the virus has only been identified in donkey abortions in China.

Prevalence of Nasal Shedding of Equid Gammaherpesviruses in Healthy Swiss Horses.

Equid Gamma herpesvirus (eGHV) infections have been reported worldwide and may be correlated with clinical signs, e.g., affecting the respiratory tract in young horses. eGHV are shed by healthy horses as well as horses with respiratory tract disease. The prevalence in healthy Swiss horses is unknown to date but this data would provide valuable information for causal diagnosis in clinical cases and formulation of biosecurity recommendations. Nasal swabs from 68 healthy horses from 12 Swiss stables and 2 stables near the Swiss border region in Germany were analyzed by panherpes nested PCR. Positive samples were sequenced. A multivariable model was used to determine if sex, age, breed, canton, or stable had a significant effect on the shedding status of each detected eGHV. Overall, the eGHV prevalence was 59% (n = 68); the prevalence for equid herpesvirus-2 (EHV-2), equid herpesvirus-5 (EHV-5) and asinine herpesvirus-5 (AHV-5) was 38%, 12% and 9%, respectively. Co-infections with multiple eGHVs were observed in 25% of the positive samples. The odds of shedding EHV-2 decreased with age (p = 0.01) whereas the odds of shedding AHV-5 increased with age (p = 0.04). Breed, sex, canton, or stable had no significant association with eGHV shedding. As EHV-2 shedding was common in healthy horses a positive PCR result must be interpreted with caution regarding the formulation of biosecurity recommendations and causal diagnosis. As EHV-5 and AHV-5 shedding was less common than EHV-2, a positive test result is more likely to be of clinical relevance. Shedding of multiple eGHV complicates the interpretation of positive test results in a horse.

Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care.

BACKGROUND: C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for "naked eye" end-point detection when testing 'real-world' clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. RESULTS: The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91-100%. CONCLUSIONS: This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing 'real-world' samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.

Epidemiology of Chlamydia psittaci infections in pregnant Thoroughbred mares and foals.

Late-term foal loss due to the traditional avian pathogen Chlamydia psittaci recently emerged as a threat to the Australian Thoroughbred industry. A longitudinal study of 14 stud farms was undertaken to better understand C. psittaci infection in pregnant mares and their foals by evaluating C. psittaci prevalence, equine herpesvirus-1 (EHV-1) co-infection, avian reservoirs, and potential risk factors. Mucosal swabs taken from 228 healthy pregnant mares and their foals were tested for C. psittaci and EHV-1 using species-specific qPCR assays. No foal loss was recorded due to either pathogen, and no mare tested positive to either C. psittaci or EHV-1. However, healthy newborn foals tested positive to both pathogens, at low levels, with 13.2% (n = 30/228) and 14.5% (n = 33/228) prevalence for C. psittaci and EHV-1, respectively. Co-infection occurred in 1.3% (n = 3/228) of foals. In avian environmental faecal samples collected from the same studs, C. psittaci was detected at 5.3% (n = 5/94). Multiple logistic regression modelling found that foals born in winter were more likely to be infected with C. psittaci (adjusted odds ratio = 15.83; P < 0.001; Confidence Interval 5.12-48.49). Being a maiden mare, absence of prophylactic vaginal suture, interventions in the last trimester and residing on a farm with prior history of C. psittaci abortion posed no higher risk to infection in the newborn. Analysis of all reported C. psittaci abortion cases (Hunter Valley, 2016-2019) revealed a dominant C. psittaci sequence type (denoted ST24) and a significant correlation with frost events (Spearmans' rho = 0.44; P = 0.002).

Outbreak of equid herpesvirus 1 abortions at the Arabian stud in Poland.

BACKGROUND: Equid herpesvirus 1 (EHV-1) infections are endemic worldwide, including Poland. Many are subclinical, but some are associated with respiratory disease, abortion, neonatal foal death, or neurological disease. We describe an outbreak of abortions in Arabian mares at a well-managed State stud farm in Poland. CASE PRESENTATION: Eight of 30 pregnant mares aborted and one gave birth to a weak foal that died within 72 h after birth. EHV-1 was isolated from all fetuses as well as from the diseased foal. All viruses belonged to the N752 variant based on the predicted open reading frame (ORF) 30 amino acid sequence. All were identical to each other and to previous EHV-1 viruses from the same stud based on the ORF68 sequence analysis. The outbreak coincided with the lapse in the routine yearly EHV-1/4 vaccinations of the mares. CONCLUSIONS: Multiple abortion due to EHV-1 infection can occur in well-managed groups of horses. Reactivation of latent EHV-1 in one of the resident mares followed by a horizontal spread was considered the most likely explanation for the outbreak. Routine vaccination is an important part of a herd-heath program.

Expressão e caracterização da glicoproteína D do herpesvírus equídeo 1 em Pichia pastoris / Expression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris

O herpesvírus equídeo 1 (EHV-1) apresenta distribuição mundial e causa graves prejuízos à equideocultura. É agente de surtos de doença respiratória, reprodutiva e neurológica, em equídeos jovens e adultos. A glicoproteína D (gD) do envelope viral é essencial para ligação e penetração em células permissivas e direcionamento do sistema imunológico do hospedeiro, induz respostas imunes humorais e celulares, sendo um antígeno apropriado para ser utilizado em vacinas e imunodiagnóstico. O objetivo deste trabalho foi expressar e caracterizar a gD do EHV-1 em Pichia pastoris para posterior utilização como antígeno em técnicas de imunodiagnóstico e formulação de vacinas recombinantes. Uma sequência de DNA que codifica uma forma truncada da gDEHV-1 foi clonada no vetor pPICZαA de expressão em P. pastoris. Obteve-se uma proteína de ~41 kDa, como esperado. A proteína apresentou glicosilação entre 4 kDa e 16 kDa, demonstrada por deglicosilação enzimática. A proteína recombinante foi caracterizada antigenicamente e imunogenicamente por Western blot, utilizando-se anticorpos policlonais equinos anti-EHV-1, e por ELISA indireto em modelo murino, demonstrando que a gD recombinante manteve epítopos similares aos da proteína nativa. Esses resultados sugerem que a gDEHV-1 é um antígeno promissor para uso como imunobiológico no controle do EHV-1.(AU)

Equine herpesvirus 1 (EHV-1) has a worldwide distribution and causes serious damage to horse breeding. It is an agent of respiratory, reproductive and neurological disease outbreaks in young and adult equids. Viral envelope glycoprotein D (gD) is essential for binding and penetration into permissive cells and targeting the host immune system, inducing humoral and cellular immune responses, and is an appropriate antigen for use in vaccines and immunodiagnostics. The objective of this work was to express in Pichia pastoris and to characterize EHV-1 gD for later use as an antigen in immunodiagnostic techniques and formulation of recombinant vaccines. A DNA sequence encoding a truncated form of gDEHV-1 has been cloned into the P. pastoris expression vector pPICZαA. A protein of ~41 kDa was obtained as expected. The protein presented glycosylation between 4 kDa and 16 kDa, demonstrated by enzymatic deglycosylation. The recombinant protein was antigenically and immunogenically characterized by Western blot using equine polyclonal anti-EHV-1 antibodies, and by indirect ELISA in a murine model, demonstrating that the recombinant gD maintained epitopes similar to those of the native protein. These results suggest that gDEHV-1 is a promising antigen for use as an immunobiological in the control of EHV-1.(AU)

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.

Molecular Surveillance of EHV-1 Strains Circulating in France during and after the Major 2009 Outbreak in Normandy Involving Respiratory Infection, Neurological Disorder, and Abortion.

Equine herpesvirus 1 (EHV-1) is an Alphaherpesvirus infecting not only horses but also other equid and non-equid mammals. It can cause respiratory distress, stillbirth and neonatal death, abortion, and neurological disease. The different forms of disease induced by EHV-1 infection can have dramatic consequences on the equine industry, and thus the virus represents a great challenge for the equine and scientific community. This report describes the progress of a major EHV-1 outbreak that took place in Normandy in 2009, during which the three forms of disease were observed. A collection of EHV-1 strains isolated in France and Belgium from 2012 to 2018 were subsequently genetically analysed in order to characterise EHV-1 strain circulation. The open reading frame 30 (ORF30) non-neuropathogenic associated mutation A2254 was the most represented among 148 samples analysed in this study. ORF30 was also sequenced for 14 strains and compared to previously published sequences. Finally, a more global phylogenetic approach was performed based on a recently described Multilocus Sequence Typing (MLST) method. French and Belgian strains were clustered with known strains isolated in United Kingdom and Ireland, with no correlation between the phylogeny and the time of collection or location. This new MLST approach could be a tool to help understand epidemics in stud farms.

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.

Molecular characterisation of equid alphaherpesvirus 1 strains isolated from aborted fetuses in Poland.

BACKGROUND: Equid alphaherpesvirus 1 (EHV-1) is one of the main infectious causative agents of abortion in mares and can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). The neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase (ORF30 D752). Another gene, ORF68 is frequently used for phylogenetic analysis of EHV-1. METHODS: 27 EHV-1 strains isolated from aborted equine fetuses in Poland, collected between 1993 and 2017, were subjected to PCR targeting the open reading frames (ORFs) 30 and 68 of the EHV-1 genome. PCR products obtained were sequenced and SNPs were analyzed and compared to sequences available in GenBank. RESULTS: None of the analyzed sequences belonged to the ORF30 D752neuropathogenic genotype: all EHV-1 belonged to the non-neuropathogenic variant N752. On the basis of ORF68 sequences, the majority of EHV-1 sequences (76.9%) cannot be assigned to any of the known groups; only six sequences (23.1%) clustered within groups II and IV. CONCLUSIONS: EHV-1 strains obtained from abortion cases belong to the non-neuropathogenic genotype. Many EHV-1 ORF68 sequences have similar SNPs to those already described in Poland, but a clear geographical distribution was not observed. A single particular ORF68 sequence type was observed in strains isolated from 2001 onwards.

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.

Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks.

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo.

Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Sensitivity and repeatability were established, and specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old fully differentiated (mature), newly seeded (immature) primary equine respiratory epithelial cell (ERECs), and equine dermal cell cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 14days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with equine herpesvirus 1 (EHV-1). The qPCR assay detected EHV-5 at stable concentrations throughout 14days in inoculated mature EREC and equine dermal cell cultures (peaking at 202 and 5861 viral genomes per 106 cellular ß actin, respectively). EHV-5 copies detected in the immature EREC cultures increased over 14days and reached levels greater than 10,000 viral genomes per 106 cellular ß actin. Moreover, EHV-5 was detected in the lymphocytes of 76% of horses and in the nasal swabs of 84% of horses experimentally infected with EHV-1 pre-inoculation with EHV-1. Post-inoculation with EHV-1, EHV-5 was detected in lymphocytes of 52% of horses while EHV-5 levels in nasal swabs were not significantly different from pre-inoculation levels. In conclusion, qPCR was a reliable technique to investigate viral load in in vivo and in vitro samples, and EHV-5 replication in equine epithelial cells may be influenced by cellular stages of differentiation.

Application of gold nanoparticle-assisted PCR for equine herpesvirus 1 diagnosis in field samples.

Equine herpesvirus 1 (EHV-1) is one of the most significant pathogens that affects equine species worldwide, causing sporadic abortion, neonatal deaths, chorioretinopathy, as well as neurological and upper respiratory tract diseases. Currently, conventional PCR targeting different genes is used widely for the molecular detection of EHV-1, but the low viral titer in some clinical samples can lead to false negative results. In this study, we aimed to assess gold nanoparticle (GNP)-assisted PCR as an inexpensive, highly efficient, and sensitive method for the detection of EHV-1, and to compare its results with conventional PCR and real-time quantitative PCR (qPCR). Out of 83 field samples, 28.9%, 26.5%, and 15.6% were EHV-1-positive by qPCR, GNP-assisted PCR and conventional PCR, respectively. All three techniques specifically target the viral glycoprotein B gene. The optimized GNP-assisted PCR showed no cross-reactivity with EHV-1-negative samples (diagnosed by qPCR). GNP-assisted PCR is a powerful new tool for EHV-1 detection and surveillance, because of its simplicity, sensitivity and specificity. It can be used as an alternative to qPCR in laboratories that cannot afford the expense of a qPCR system.