The effect of maternal immunity on the equine gammaherpesvirus type 2 and 5 viral load and antibody response.

Two types of gammaherpesviruses (γEHV) are known to infect horses, EHV-2 and EHV-5. Foals become infected early in life, probably via the upper respiratory tract, despite maternal antibodies. In this study, we analyzed samples from a herd of mares and their foals. The foals were followed from birth to 22 months of age and the dams during the first 6 months postpartum. Blood and nasal swab samples were taken regularly for evaluation of antibody responses, virus isolation and viral load by qPCR. EHV-2 was isolated on day 5, and EHV-5 on day 12, earlier than previously reported. γEHV specific antibodies were not detectable in serum of foals before colostrum intake but peaked a few days after colostrum. Overall, EHV-2 viral load peaked in nasal swab at three to four months of age, paralleled with decline in maternal antibodies, but EHV-5 viral load did not peak until month 12. Maternal antibodies had a notable effect on the viral load and induction of endogenous antibody production. Foals were grouped in two groups depending on the mare's γEHV specific total IgG levels in serum at birth, group-high and group-low. Group-high had higher levels of maternal γEHV specific total IgG and IgG4/7 for the first 3 months, but when the endogenous production had superseded maternal antibodies, group-low was higher. The maternal antibodies had an effect on the γEHV viral load. Group-low peaked in EHV-2 viral load one month earlier than group-high. These effects were more evident for EHV-5, as there were seven months between the viral load peaks for the groups. The study provides information on how maternal antibody transfer affects γEHV shedding and antibody production in offspring. It also extends our knowledge on the occurrence of EHV-2 and EHV-5 infection in foals during the first two years of life.

Neonatal Immunization with a Single IL-4/Antigen Dose Induces Increased Antibody Responses after Challenge Infection with Equine Herpesvirus Type 1 (EHV-1) at Weanling Age.

Neonatal foals respond poorly to conventional vaccines. These vaccines typically target T-helper (Th) cell dependent B-cell activation. However, Th2-cell immunity is impaired in foals during the first three months of life. In contrast, neonatal basophils are potent interleukin-4 (IL-4) producers. The purpose of this study was to develop a novel vaccine triggering the natural capacity of neonatal basophils to secrete IL-4 and to evaluate if vaccination resulted in B-cell activation and antibody production against EHV-1 glycoprotein C (gC). Neonatal vaccination was performed by oral biotinylated IgE (IgE-bio) treatment at birth followed by intramuscular injection of a single dose of streptavidin-conjugated gC/IL-4 fusion protein (Sav-gC/IL-4) for crosslinking of receptor-bound IgE-bio (group 1). Neonates in group 2 received the intramuscular Sav-gC/IL-4 vaccine only. Group 3 remained non-vaccinated at birth. After vaccination, gC antibody production was not detectable. The ability of the vaccine to induce protection was evaluated by an EHV-1 challenge infection after weaning at 7 months of age. Groups 1 and 2 responded to EHV-1 infection with an earlier onset and overall significantly increased anti-gC serum antibody responses compared to control group 3. In addition, group 1 weanlings had a decreased initial fever peak after infection indicating partial protection from EHV-1 infection. This suggested that the neonatal vaccination induced a memory B-cell response at birth that was recalled at weanling age after EHV-1 challenge. In conclusion, early stimulation of neonatal immunity via the innate arm of the immune system can induce partial protection and increased antibody responses against EHV-1.

Establishment and characterization of fetal equine kidney and lung cells with extended lifespan. Susceptibility to equine gammaherpesvirus infection and transfection efficiency.

Due to the slow growth of equine gammaherpesviruses, isolation of these viruses requires cells that can be propagated long term and show clear cytopathy following infection. Equine cell lines with extended lifespan were established from primary cells originating from equine fetal kidney and lung by transfecting the cells with the retroviral vector LXSN116E6E7 containing the human papilloma virus oncogenes 16 E6 and E7. The transfected equine kidney cell line and equine lung cell line can be propagated for more than 40 passages, whereas the corresponding primary cells only for 10-12 passages. The primary cells and the derived cell lines can be infected with equine gammaherpesvirus 2 (EHV-2) with similar efficiency. However EHV-5 can be grown to a substantially higher titer in the kidney cell line than their primary counterpart, with cytopathic effect visible three days earlier than in the primary cells. Due to rapid cell growth the lung cell line is difficult to use for virus production. The kidney cell line was four times more susceptible to transfection as compared to the primary kidney cells. On the other hand no difference was between the lung cell line and the primary lung cells in transfection efficiency. The cell lines can be a valuable tool for investigating gammaherpesviruses, and possibly other viruses infecting horses.

Genetic diversity of equine gammaherpesviruses (γ-EHV) and isolation of a syncytium forming EHV-2 strain from a horse in Iceland.

The horse population in Iceland is a special breed, isolated from other equines for at least one thousand years. This provides an exceptional opportunity to investigate old and new pathogens in a genetically closed herd. Both types of equine gammaherpesviruses, EHV-2 and EHV-5, are common in Iceland. Genetic variation was examined by sequencing four genes, glycoprotein B (gB), glycoprotein H (gH), DNA polymerase and DNA terminase for 12 Icelandic and seven foreign EHV-2 strains. One Icelandic virus isolate, gEHV-Dv, induced syncytium formation, an uncharacteristic cytopathy for EHV-2 in equine kidney cells. When sequenced, the glycoprotein genes were different from both EHV-2 and EHV-5, but the polymerase and terminase genes had 98-99% identity to EHV-2. Therefore the gEHV-Dv strain can be considered a variant of EHV-2. Substantial genetic variability was seen within the EHV-2 glycoprotein genes but limited in the polymerase and terminase genes. The Icelandic EHV-2 strains do not seem to differ phylogenetically from the foreign viruses, despite isolation for over a thousand years.

Generation of equine TSLP-specific antibodies and their use for detection of TSLP produced by equine keratinocytes and leukocytes.

Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant eqTSLP protein and generate TSLP-specific antibodies. EqTSLP was cloned from a skin biopsy sample from a horse with chronic urticaria and eqTSLP protein was expressed in E.coli and in mammalian cells. Recombinant proteins were designed to include C-terminal Histag, which allowed subsequent purification and detection by Histag-specific Ab. Polyclonal and monoclonal eqTSLP-specific Ab were generated after immunization of mice with E.coli-expressed TSLP. Their specificity was tested by western blotting and ELISA. In addition, a commercially available polyclonal human TSLP-specific antibody was tested for cross-reactivity with eqTSLP. Expression of TSLP protein was confirmed by western blotting using Histag-specific Ab. E.coli-expressed TSLP appears as a band of ∼13 kDa, whereas mammalian cell-expressed TSLP showed several bands at 20-25 kDa, probably representing several glycosylation forms. Polyclonal and monoclonal antibodies generated in this study, as well as commercially available human TSLP-specific Ab reacted with both E.coli- and mammalian cell-expressed TSLP in western blotting and ELISA. A capture ELISA was established to quantitate TSLP in cell supernatants and validated using supernatants from primary equine keratinocytes and peripheral blood leukocytes (PBL). Increased TSLP concentrations were found after stimulation of keratinocytes with PMA+ionomycine and with Culicoides extract. Similarly, increased TSLP concentrations were detected in PBL after stimulation with ConA, Culicoides extract, or IgE cross-linking. In conclusion, recombinant TSLP proteins and TSLP-specific antibodies produced in this study will allow further studies of the role of TSLP in equine allergic diseases.

Skin-infiltrating T cells and cytokine expression in Icelandic horses affected with insect bite hypersensitivity: a possible role for regulatory T cells.

Equine insect bite hypersensitivity (IBH) is a seasonally recurrent, pruritic skin disorder caused by an IgE-mediated reaction to salivary proteins of biting flies, predominantly of the genus Culicoides. The aim of this study was to define T cell subsets and cytokine profile in the skin of IBH-affected Icelandic horses with particular focus on the balance between T helper (Th) 1, Th2 and T regulatory (Treg) cells. Distribution and number of CD4+, CD8+ and Forkhead box P3 (FoxP3)+ T cells were characterized by immunohistochemical staining in lesional and non-lesional skin of moderately and severely IBH-affected horses (n=14) and in the skin of healthy control horses (n=10). Using real-time quantitative reverse transcription-polymerase chain reaction, mRNA expression levels of Th2 cytokines (Interleukin (IL)-4, IL-5, IL-13), Th1 cytokines (Interferon-γ), regulatory cytokines (Transforming Growth Factor ß1, IL-10) and the Treg transcription factor FoxP3 were measured in skin and blood samples. Furthermore, Culicoides nubeculosus specific serum IgE levels were assessed. Lesions of IBH-affected horses contained significantly higher numbers of CD4+ cells than skin of healthy control horses. Furthermore, the total number of T cells (CD4+ and CD8+) was significantly increased in lesional compared to non-lesional skin and there was a tendency (p=0.07) for higher numbers of CD4+ cells in lesional compared to non-lesional skin. While the number of FoxP3+ T cells did not differ significantly between the groups, the ratio of Foxp3 to CD4+ cells was significantly lower in lesions of severely IBH-affected horses than in moderately affected or control horses. Interestingly, differences in FoxP3 expression were more striking at the mRNA level. FoxP3 mRNA levels were significantly reduced in lesional skin, compared both to non-lesional and to healthy skin and were also significantly lower in non-lesional compared to healthy skin. Expression levels of IL-13, but not IL-4 or IL-5, were significantly elevated in lesional and non-lesional skin of IBH-affected horses. IL-10 levels were lower in lesional compared to non-lesional skin (p=0.06) and also lower (p=0.06) in the blood of IBH-affected than of healthy horses. No significant changes were observed regarding blood expression levels of Th1 and Th2 cytokines or FoxP3. Finally, IBH-affected horses had significantly higher Culicoides nubeculosus specific serum IgE levels than control horses. The presented data suggest that an imbalance between Th2 and Treg cells is a characteristic feature in IBH. Treatment strategies for IBH should thus aim at restoring the balance between Th2 and Treg cells.

Isolation and partial sequencing of Equid herpesvirus 5 from a horse in Iceland.

Horses are hosts to 2 types of gammaherpesviruses, Equid herpesvirus 2 and 5 (EHV-2 and EHV-5, respectively). Both EHV-2 and EHV-5 are common in horses in Iceland. An Icelandic EHV-5 isolate was recovered by sequential culture in primary fetal horse kidney and rabbit kidney cells. Glycoprotein B, glycoprotein H, and DNA terminase genes of the isolate were fully sequenced, and the DNA polymerase gene was partly sequenced. To date, the glycoprotein B gene of EHV-5 was the only gene that has been reported to be completely sequenced in addition to small parts of the glycoprotein H, DNA polymerase, and DNA terminase genes. The present report, therefore, is a significant addition to previously reported EHV-5 sequences.

Immune response against equine gammaherpesvirus in Icelandic horses.

Horses are hosts to two types of gammaherpesviruses, equine herpes virus (EHV) 2 and 5. While EHV-2 is ubiquitous in adult horses, EHV-5 has been less frequently described. Due to strong serological cross-reactivity, EHV-2 and -5 cannot be discriminated in broad spectrum antibody tests and are thus commonly referred to as gamma-EHV. Total IgG and IgG subclass response against gamma-EHV were determined in serum from 41 healthy Icelandic horses, thereof 20 adults, 10 foals aged 10 months, and 11 foals aged 1-4 months. Additionally, in 10 of the adult horses, interferon (IFN)-gamma and interleukin (IL)-4 expression were measured by real-time PCR in white blood cells upon in vitro stimulation with EHV-2. With the exception of one orphan foal, all tested individuals were seropositive for gamma-EHV. All but one adult had high titer of EHV-specific IgG4/7 (IgGb) in combination with much lower titer of IgG1 (IgGa) and IgG3/5 (IgG(T)), indicating a stabilized response. IgG titer and subclasses in the foals showed considerably more variation, possibly dependant on maternal antibodies and/or recent infection. In all the 10 horses tested for cytokine expression, IFN-gamma production exceeds production of IL-4. These results indicate that equine gammaherpesvirus infection is characterized by an induction of IgG1, IgG4/7 and IgG3/5 with prevailing IgG4/7 and cytokine profile dominated by IFN-gamma. To our knowledge, this is the first report on the cytokine and IgG subclass response against gamma-EHV in horses.

Study of equid herpesviruses 2 and 5 in Iceland with a type-specific polymerase chain reaction.

The horse population in Iceland is a special breed, isolated from other horses for at least 1000 years. This provides an exceptional opportunity to investigate old and new pathogens in an inbred herd with few infectious diseases. We have developed a high sensitivity semi-nested PCR to study equid gammaherpesviruses 2 and 5 (EHV-2 and 5) in Iceland. The first PCR is group specific, the second type-specific, targeting a 113bp sequence in the glyB gene. DNA isolated from white blood cells and 18 different organs was tested for the presence of EHV-2 and 5. This was done in adult horses and foals, healthy and with various enteric infections. Both virus types were easily detected in all types of organs tested or EHV-2 in 79% cases and EHV-5 in 63%. In DNA from PBMC or buffy-coat EHV-2 was found in 20% cases and EHV-5 in 10%, all except one positive were foals. Co-culture of PBMC on fetal horse kidney cells was efficient for detecting EHV-2 but not for EHV-5. We verify here for the first time infections with EHV-2 and 5 in horses in Iceland and show that both viruses are common.

Mutational analysis of a principal neutralization domain of visna/maedi virus envelope glycoprotein.

We have shown previously that a type-specific neutralization domain is located within a 39 aa sequence in the fourth variable domain of gp135 in visna/maedi virus. We now show that neutralizing antibodies detected early in infection are directed to this epitope, suggesting an immunodominant nature of this domain. Ten antigenic variants were previously analysed for mutations in this region, and all but one were found to be mutated. To assess the importance of these mutations in replication and neutralization, we reconstructed several of the mutations in an infectious molecular clone and tested the resulting viruses for neutralization phenotype and replication. Mutation of a conserved cysteine was shown to alter the neutralization epitope, whilst the replication kinetics in macrophages were unchanged. Mutations modulating potential glycosylation sites were found in seven of the ten antigenic variants. A frequently occurring mutation, removing a potential glycosylation site, had no effect on its own on the neutralization phenotype of the virus. However, adding an extra potential glycosylation site in the region resulted in antigenic escape. The results indicate that the conserved cysteine plays a role in the structure of the epitope and that glycosylation may shield the principal neutralization site.

Vaccination of sheep with Maedi-visna virus gag gene and protein, beneficial or harmful?

In spite of intense efforts no vaccine is yet available that protects against lentiviral infections. Sheep were immunised eight times over a period of 2.5 years with the maedi-visna (MVV) gag gene on two different vectors, 2 sheep with VR1012-gag-CTE and 2 sheep with pcDNA3.1-gag-CTE. All sheep responded to some of the mature MVV Gag proteins in Western blot (WB). Three of them responded to the virus in lymphocyte proliferation test. The sheep received a boost with recombinant Gag protein resulting in elevated antibody response. However, when they were challenged intratracheally with MVV they all became immediately infected as judged by a strong rise in antibody titer and virus isolation from blood. It is therefore clear that the vaccination gave no protection. It is even possible that it facilitated infectivity since virus was isolated earlier from all the vaccinated sheep than from any of the unvaccinated sheep infected in the same way with the same dose.

Simultaneous mutations in CA and Vif of Maedi-Visna virus cause attenuated replication in macrophages and reduced infectivity in vivo.

Maedi-visna virus (MVV) is a lentivirus of sheep sharing several key features with the primate lentiviruses. The virus causes slowly progressive diseases, mainly in the lungs and the central nervous system of sheep. Here, we investigate the molecular basis for the differential growth phenotypes of two MVV isolates. One of the isolates, KV1772, replicates well in a number of cell lines and is highly pathogenic in sheep. The second isolate, KS1, no longer grows on macrophages or causes disease. The two virus isolates differ by 129 nucleotide substitutions and two deletions of 3 and 15 nucleotides in the env gene. To determine the molecular nature of the lesions responsible for the restrictive growth phenotype, chimeric viruses were constructed and used to map the phenotype. An L120R mutation in the CA domain, together with a P205S mutation in Vif (but neither alone), could fully convert KV1772 to the restrictive growth phenotype. These results suggest a functional interaction between CA and Vif in MVV replication, a property that may relate to the innate antiretroviral defense mechanisms in sheep.