Abstract. 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.Key words: Equid herpesvirus 5; gammaherpesviruses; horses; sequencing; virus isolation.Five herpesviruses are known to infect horses; 2 of them, Equid herpesvirus 2 (EHV-2) and Equid herpesvirus 5 (EHV-5), are closely related 1,7,27 (order Herpesvirales, family Herpesviridae, subfamily Gammaherpesvirinae, genus Percavirus). Foals become infected with EHV-2 and EHV-5 at 1-6 months of age, probably via the upper respiratory tract. 3,15,21 After the primary infection, the virus establishes a latent infection, most likely in the case of EHV-2 in B lymphocytes. 10 Equid herpesvirus 2 has been associated with keratoconjunctivitis, respiratory disease, pneumonia, pharyngitis, fever, enlarged lymph nodes, and lack of appetite. 2,6,8,11,18,20,25 Equid herpesvirus 5 was recently associated with multinodular pulmonary fibrosis. 29 Equid herpesvirus 2 has been detected in the majority of horses worldwide, and EHV-5 is also widespread but less common. 5,9,22,24 The 2 viruses have a strong serologic cross reactivity 1,23 and may coexist in the same horse. 7 Less is known about EHV-5 than about EHV-2, and only a few isolates have been described. 4 The 2 viruses have been shown to be common in Iceland. 28 The current report describes the isolation of EHV-5 from peripheral blood mononuclear cells from a 9-year-old mare (isolate BB5). The peripheral blood mononuclear cells from 2 Listeria monocytogenes-infected horses 16 were cocultured with primary fetal equine kidney cells (EqFKC) as previously described. a-c,28 Two isolates, BB5 and BB11, were shown, with type-specific polymerase chain reaction (PCR), to be mixtures of EHV-2 and EHV-5.Plaque purification of EHV-5 was attempted in EqFKCs but was not successful until the BB5 and BB11 isolates were alternately passaged in EqFKCs and rabbit kidney cells (RK-13), d which have previously been used for isolation of EHV-5. 4,12 The RK-13 cells were infected with BB5 or BB11 supernatant from EqFKCs and incubated for 8 days. The supernatant from the RK-13 cells was subsequently harvested and used to infect fresh EqFKCs that were incubated until they showed almost 100% cytopathology. Fresh RK-13 cells were then infected as before with this EqFKCs supernatant and incubated for 8 days when the supernatant was harvested and plaque purification carried out in EqFKCs as pre...
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.
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.
Equine coital exanthema (ECE) caused by equid alphaherpesvirus 3 (EHV-3) is a contagious venereal disease. It is characterized by the formation of papules, vesicles, pustules and ulcers on the external genitals of both mares and stallions. The Icelandic horse is the only breed in Iceland and has lived isolated in the country for over 1000 years. Three types of equine herpesviruses (EHV) have been found in Iceland, EHV-4, EHV-2 and EHV-5, while EHV-1 has never been detected. Symptoms resembling ECE have previous been observed in horses in Iceland, arousing suspicion of EHV-3 infection, but this has never been confirmed using virological methods. Samples were collected from a mare with papules on the vulva and inoculated in primary equine kidney cells. Cytopathic effects developed as rounded cells and syncytial formation. Polymerase chain reaction and sequencing of the partial glycoprotein G and DNA polymerase genes identified the isolated virus as EHV-3. On the basis of the findings, EHV-3 infection was verified for the first time in the native Icelandic horse population.
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