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A single-dilution, sensitive and specific monoclonal antibody-based blocking enzyme-linked immunosorbent assay (B-ELISA) was developed as an alternative to the cumbersome virus neutralization test (VNT) for detection of equine herpesvirus-1 (EHV-1) antibodies. JavaScript is currently disabled, this site works much better if you enable JavaScript in your browser. Institut fur Virologie, Robert von Ostertag-Haus, Zentrum fur Infektionsmedizin, Freie Universitat Berlin, Robert-von-Ostertag-Str.
Amino acid sequence alignment of Equine Herpesvirus Type 1 and Type 4 (EHV-1 and EHV-4) glycoprotein B (gB). In the case of EHV-4_gB1, however, significantly reduced plaque sizes were seen compared to parental and revertant viruses (Figure 5b).
Neutralizing monoclonal antibodies (1H6 and 9C6) raised against EHV-1 (Hisar-90-7 strain) and sera from 70 horses (30 known negative and 40 known positive for EHV-1 antibodies by VNT) were used for standardization of the B-ELISA. To evaluate whether functional differences exist between gB1 and gB4, the corresponding genes were exchanged between the two viruses.
IntroductionGlycoprotein B (gB) is a type 1 transmembrane protein and represents a highly conserved class III fusion protein present in members of the Herpesviridae family [1].
The putative integrin-binding motif tyrosine-glycine-leucine (YGL) present in the extracellular domains of both gB1 and gB4 (red frame).
YGL is also present in the VP4 spike protein of rotaviruses where it mediates cell entry [36]. The two unique regions (UL and US) of the EHV-1 genome as well as the terminal and internal repeat sequences (TRS and IRS) are shown, as is the inserted mini-F cassette.
Cell lysates were prepared from infected equine dermal (ED) cells and proteins were separated under reducing conditions by sodium dodecyl sulfate (SDS)-10%-polyacrylamide gel electrophoresis (PAGE). Virus Growth in CultureNo significant differences were observed for any of the EHV-1 recombinants when compared to the parental virus (Figure 5a,c,d). ED cells were infected with the respective viruses at a multiplicity of infection (MOI) of 0.01. Similarly, EHV-4_gB1 also showed a significantly reduced growth rate at several time points as evidenced by the growth kinetics (Figure 5e). Using a single serum dilution of 1:250 in B-ELISA, 100% specificity was obtained with both monoclonal antibodies (Mabs) in comparison to VNT. The gB4-containing-EHV-1 (EHV-1_gB4) recombinant virus was analyzed for growth in culture, cell tropism, and cell entry rivaling no significant differences when compared to parental virus. In a recent study, a similar integrin binding motif, leucine-aspartic acid-isoleucine (LDI), present in EHV-1 gH and interacting with cellular ?4?1 integrins, has been implicated in determining the entry pathway taken by EHV-1 in equine cells [32]. PCR products of the kanamycin resistance gene (KanR) were transformed into GS1783 containing the EHV-1 bacterial artificial chromosomes (BACs) by electroporation and the first step of the Red recombination method ensued. We therefore concluded that the growth properties of recombinant EHV-1 in culture were not significantly affected by the exchange with EHV-4 gB. Similarly, the sensitivity of the B-ELISA was 92.5% and 100% with 1H6 and 9C6 Mabs, respectively.
We also disrupted a potential integrin-binding motif, which did not affect the function of gB in culture. In Herpes simplex virus type 1 (HSV-1), gB has been shown to bind several cellular receptors to facilitate viral entry; however, there is no data available on the role of equine herpesvirus type 1 (EHV-1) gB in receptor binding [1]. Sequences were aligned using Vector NTI software (version 9, Invitrogen, Carlsbad, CA, USA, 2004). EHV-1, EHV-4 and related recombinant virus proteins with an apparent molecular weight of approximately 130 and 76 kD were detected that are not present in mock-infected cells.
In contrast, Vero cells transfected with parental EHV-4 DNA showed syncytium formation after 48 h (Figure 4c). After 48 h of incubation, cells were inspected with an epifluorescent microscope (Zeiss Axiovert, Jena, Germany) and images were taken with a CCD camera (Zeiss Axiocam, Jena, Germany). The bar represents 100 ?m and the white frames contain magnified inserts of the selected areas. Epitopes of glycoprotein G of equine herpes virus 4 and 1 located near the C termini elicit type specific antibody responses in the natural host. In contrast, a significant reduction of plaque sizes and growth kinetics of gB1-containing-EHV-4 (EHV-4_gB1) was evident when compared to parental EHV-4 and revertant viruses.
HSV-1 gB can bind to the paired immunoglobulin-like type 2 receptor (PILR?) to trigger viral fusion in the presence of gD [10]. Rabbit anti-?-actin antibody was used as a loading control (molecular weight of approximately 42 kDa). CHO-K1, CHO-A, CHO-B and CHO-C, and RK13 cells were highly resistant and MDCK virtually resistant to parental and recombinant EHV-4 infection. B-ELISA detected a more than 3-fold rise in antibody titres in paired serum samples collected from mares aborting owing to EHV-1 infection.
In addition, CrFK were highly resistant and Vero cells virtually resistant to parental and recombinant EHV-1 infection. Mab 9C6 was chosen for testing 231 field sera from apparently healthy vaccinated and non-vaccinated horses from organized breeding farms belonging to 11 Indian states, and from Bhutan, by B-ELISA and VNT. Alternatively, gB4 might have an additional function, required for EHV-4 replication, which is not fulfilled by gB1.
There was very good agreement between the results obtained by both VNT and B-ELISA (? = 0.9438). In conclusion, our results show that the exchange of gB between EHV-1 and EHV-4 is possible, but results in a significant attenuation of virus growth in the case of EHV-4_gB1.
Later, it was shown that, like gB of other alphaherpesviruses, EHV-1 gB is essential for viral growth and direct cell-to-cell spread.
Of 231 field sera, 144 samples were negative for EHV-1 antibodies by both VNT and B-ELISA and 81 were positive by both tests. The generation of stable recombinant viruses is a valuable tool to address viral entry in a comparative fashion and investigate this aspect of virus replication further.
This was deduced from the fact that only single infected cells, but no viral plaques, were observed when EHV-1?gB was used to infect non-complementing cell lines.


For single step growth kinetics of EHV-1 recombinant viruses, ED cells were infected at an MOI of 1 (c,d), followed by citrate treatment (pH = 3) to remove remaining extra-cellular virions. A strong indication for its role as a fusogen was also reported; when viral titers of a gB-deficient EHV-1 virus could partly be restored by adding polyethylene glycol to induce fusion [3]. Infected cells (c) and supernatants (d) were separately collected and virus titers were determined at the indicated times post-infection (p.i). Detection of equine herpes virus-1 antigen and the specific antibody by enzyme-linked immunosorbent assay. However, there is no data available on the function of EHV-4 gB, the close relative of EHV-1.EHV-4 (145 kbp) and EHV-1 (150 kbp) both contain 76 unique and highly similar genes. The Mab (9C6)-based B-ELISA was found to be a suitable alternative to VNT for screening large numbers of field sera and enabled confirmatory EHV-1 serodiagnosis. Nucleic acid identity between EHV-4 and EHV-1 genes in general is above 80%, indicating that they are functionally closely related [22,23,24].
Por medio de una PCR semianidada se amplificaron los genes que codifican por las glicoproteína Hs (gH) de HVE-1 y B (gB) de HVE-4. However, despite this high genetic similarity, the disease outcomes after infection differ substantially. EHV-4 only induces mild symptoms that are usually limited to upper respiratory tract infections associated with fever and general malaise [25,26]. EHV-1 and EHV-4 gB genes were amplified by PCR using primers P1 and P2 or P3 and P4 (Table 1). Infected cells and supernatants were collected and virus titers were determined at the indicated times p.i. While also causing respiratory disease, EHV-1 induces more severe clinical disease that includes abortion in pregnant mares [27,28,29] and the so-called EHV-1 myeloencephalopathy (EHM) [30,31]. Virus Growth AssaysTo determine differences in viral replication in culture, single step growth kinetics for EHV-1 and multi-step growth kinetics for EHV-4 were conducted as described before [34]. Correct amplification and insertion were confirmed by Sanger sequencing (LGC Genomics, Berlin, Germany). Briefly, confluent ED cells were infected with a multiplicity of infection (MOI) of 1 in case of EHV-1 parental virus, EHV-1_gB4 and EHV-1 revertant.
More specifically, EHV-1 enters equine epithelial cells via direct fusion at the plasma membrane, while EHV-4 does so via an endocytic pathway [32].The close genetic relatedness allowed us to exchange essential genes between EHV-1 and EHV-4 and to evaluate the effects both in vitro and in vivo in a way not possible for other members of the subfamily since EHV-1 and EHV-4 naturally infect the same host. Enzyme-linked immunosorbent assay for the detection of antibodies to equid herpes virus type-1 (EHV-1).
We have been interested in exchanging glycoproteins that are part of the cell entry complex between EHV-1 and EHV-4 to further elucidate the process of virus entry [32,33,34,35]. So far, gD was found to play an essential role in determining the cellular tropism of EHV-1 and EHV-4 in culture [33].
Manual of Standards for Diagnostic Tests and Vaccines, (Office International Des Epizooties, Paris), 426-433Reed, L.J.
We were interested in exchanging gB to uncover possible functional differences between the two viruses, thereby further elucidating the role of gB in tropism and pathogenicity. Soon after this takes place a primary cell associated viremia to peripheral blood mononuclear cells (PBMC, mainly on B and T lymphocytes), which allows the virus to reach other organic systems and production of abortions in the last third of gestation, neonatal foal death and neurological syndromes. The obtained values were normalized and compared to the values of parental viruses, which were set to 100%. Because the presence of antibodies for EHV-1 and EHV-4 in plasma and serum of horses of two departments of Colombia was demonstrated, the objective of the present study as to demonstrate the presence of the viral genome in PBMC from horses diagnosed seropositive for EHV-1 and EHV-4, and in trigeminal ganglion of equines from a slaughterhouse of the Department of Antioquia. Three independent experiments were used to calculate average plaque sizes and standard deviations. By means of a semi-nested PCR, the gene codifying for glycoprotein H (gH) of EHV-1 and gB of EHV-4 were amplified. The characterization of neutralizing and non-neutralizing monoclonal antibodies against equid herpesvirus type-1.
With the aim of assessing whether the circulating strain in the department of Antioquia had a neuropathogenic potential, we amplified and sent to sequencing the gene that encodes the viral DNA polymerase, which could has a mutation that has been associated with neuropathogenic potential. The set of our results confirms that infection by EHV is present in the State of Antioquia, Colombia, and that there are equines latently infected which can be a source of infection for other susceptible horses. Cytopathic expression and growth of equine herpes virus-1 recovered from aborted fetuses in three cell-culture systems.
Flow CytometryOne million PBMC were incubated with EHV-1 and the engineered recombinant viruses at an MOI of 1 for 48 h at 37 °C.
Detection of herpes virus-1 specific antibodies by enzyme-linked immunosorbent assay (ELISA) in equines. After incubation, the percentage of infected cells was determined by measuring eGFP expression with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Comparison of enzyme-linked immunosorbent assay, virus neutralization and complement fixation tests for measurement of antibodies against equine herpes virus-1 infection in equine sera.
Normally distributed datasets, determined with the Shapiro–Wilks test, were analyzed with one-way ANOVA. Datasets that were not normally distributed were analyzed with Kruskal–Wallis one-way analysis of variance for two or more samples that are independent or the Friedman test for repeated measures.
Neutralizing and complement-fixing monoclonal antibodies as an aid to the diagnosis of equine herpesvirus-1 infection. La infección in vivo de MNSP fue descrita por primera vez en 1983 por Scott et a, (33). Differentiation of Indian isolates of equine herpes virus (EHV-1) by restriction endonuclease digestion. Equine herpes viruses: on the differentiation of respiratory from foetal strains of type-1.
Por el contrario, estudios con HVE-4 demuestran que este no establece latencia en linfocitos de sangre periférica (6, 28, 48). Nucleic acid pattern of an Indian isolate of equine herpes virus-1 using restriction endonuclease.


Wade (eds), Equine Infectious Diseases VI, (Cambridge University Press, Cambridge), 121Van de Moer, A., Rice, M.
A type specific conformational epitope on the nucleocapsid of equid herpes virus-1 and its use in diagnosis.
Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV-1) and equine herpes virus type 4 (EHV-4). Para amplificar el ORF 68 y ORF 30 de HVE-1, se realizó una PCR siguiendo las condiciones reportadas por Nugent et al (26).
Los dos ORF se amplificaron de todas las muestras que fueron positivas para la gH del HVE-1. Seis muestras de DNA aisladas de MNSP fueron enviadas a secuenciar a Macrogen® (Seul, Korea), siguiendo todas las recomendaciones establecidas por dicha casa comercial. Por su parte, la figura 4 muestra que en 22 del los 38 ganglios (58%) se amplificó el gen que codifica para la gH de HVE-1 y en 18 (48%) el gen para la gB de HVE-4. Infortunadamente, las secuencias obtenidas con los amplificados del ORF68 de HVE-1, no fueron suficientes para poder determinar el posible origen del virus que circula en la región de Antioquia, debido a que las secuencias legibles en el electroferograma fueron demasiado cortas. Rafael Villalobos, del sistema de vigilancia epidemiológica de encefalitis virales del Instituto Colombiano Agropecuario ICA-CEISA). En su conjunto, estos resultados sugieren que existen individuos infectados por los HVE-1 y HVE-4, los cuales pueden actuar como reservorios de infección para otros equinos susceptibles. Epidemic disease caused by Equine Herpesvirus-1: recommendation for prevention and control.
Quantification by real-time PCR of the magnitude and duration of leukocyte-associated viraemia in horses infected with neuropathogenic versus non-neuropathogenic strains of Equid herpesvirus-1. Occurrence of equine herpes virus type-1 (EHV-1)-specific DNA sequences in peripheral blood leukocytes of horses.
The detection of latency-associated transcripts of equine herpesvirus 1 in ganglionic neurons. Equine herpesvirus 4 DNA in trigeminal ganglia of naturally infected horses detected by direct in situ PCR. Latency-associated transcripts of equine herpesvirus type 4 in trigeminal ganglia of naturally infected horses. Detection of latency-associated transcripts of equid herpesvirus 1 in equine leukocytes but not in trigeminal ganglia. Infectious center assay of intracellular virus and infective virus titer for equine mononuclear cells infected in vivo and in vitro with equine herpesviruses. Endothelial cell infection and thrombosis in paralysis caused by equid herpesvirus-1: equine stroke. A polymerase chain reaction for detection of equine herpesvirus-1 in routine diagnostic submissions of tissues from aborted foetuses.
Argentine strain of equine herpesvirus 1 isolated from an aborted foetus shows low virulence in mouse respiratory and abortion models.
Pathogenesis of equine herpesvirus-1 in specific pathogen-free foals: primary and secondary infections and reactivation.
MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Distribution of equid herpesvirus-1 (EHV-1) in respiratory tract of ponies: implications for vaccination strategies. Distribution of equid herpesvirus-1 (EHV-1) in respiratory tract associated lymphoid tissue: implications for cellular immunity. Clinical, serological and virological characteristics of an outbreak of paresis and neonatal foal disease due to equine herpesvirus-1 on a stud farm. Serological and virological investigations of an equid herpesvirus 1 (EHV1) abortion storm on a stud farm in 1985. Abortigenic and neurological disease caused by experimental infection with equid herpesvirus-1. The nucleotide sequence of an equine herpesvirus 4 gene homologue of the herpes simplex virus 1 glycoprotein H gene. Analysis of equid herpesvirus type 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus non-neuropathogenic disease outbreaks.
Equine herpesvirus-4 kinetics in peripheral blood leukocytes and nasopharyngeal secretions in foals using quantitative real-time TaqMan PCR. Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4. In vivo harboring of equine herpesvirus-1 in leukocyte populations and subpopulations and their quantitation from experimentally infected ponies. The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in the horse. Infection of endothelial cells with equine herpesvirus-1 (EHV-1) occurs where there is activation of putative adhesion molecules: a mechanism for transfer of virus. A study of the pathogenesis of equid herpesvirus-1 (EHV-1) abortion by DNA in-situ hybridization.
Replication of equine herpesvirus type 1 in freshly isolated equine peripheral blood mononuclear cells and changes in susceptibility following mitogen stimulation. Mitogen stimulation favours replication of equine herpesvirus 1 in equine blood mononuclear cells by inducing cell proliferation and formation of close intercellular contacts. Increased susceptibility of peripheral blood mononuclear cells to equine herpes virus type 1 infection upon mitogen stimulation: a role of the cell cycle and of cell-to-cell transmission of the virus.
Latent equid herpesviruses 1 and 4: detection and distinction using the polymerase chain reaction and co-cultivation from lymphoid tissues.



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