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.

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.

Global identification of three major genotypes of varicella-zoster virus: longitudinal clustering and strategies for genotyping.

By analysis of a single, variable, and short DNA sequence of 447 bp located within open reading frame 22 (ORF22), we discriminated three major varicella-zoster virus (VZV) genotypes. VZV isolates from all six inhabited continents that showed nearly complete homology to ORF22 of the European reference strain Dumas were assigned to the European (E) genotype. All Japanese isolates, defined as the Japanese (J) genotype, were identical in the respective genomic region and proved the most divergent from the E strains, carrying four distinct variations. The remaining isolates carried a combination of E- and J-specific variations in the target sequence and thus were collectively termed the mosaic (M) genotype. Three hundred twenty-six isolates collected in 27 countries were genotyped. A distinctive longitudinal distribution of VZV genotypes supports this approach. Among 111 isolates collected from European patients, 96.4% were genotype E. Consistent with this observation, approximately 80% of the VZV strains from the United States were also genotype E. Similarly, genotype E viruses were dominant in the Asian part of Russia and in eastern Australia. M genotype viruses were strongly dominant in tropical regions of Africa, Indochina, and Central America, and they were common in western Australia. However, genotype M viruses were also identified as a minority in several countries worldwide. Two major intertypic variations of genotype M strains were identified, suggesting that the M genotype can be further differentiated into subgenotypes. These data highlight the direction for future VZV genotyping efforts. This approach provides the first simple genotyping method for VZV strains in clinical samples.

Polymerase chain reaction for laboratory diagnosis of orf virus infections.

BACKGROUND: The orf virus of sheep and goats is one of several zoonotic parapoxviruses. In the ovine/caprine host it causes contagious ecthyma (contagious pustular dermatitis, scabby mouth), but in humans it normally causes solitary or clustered orf lesions, typically on hands, arms or face. In addition to disease in the animals, the virus can be quite a nuisance as an occupational hazard in farmers and butchers. Clinical diagnosis is often possible, but laboratory diagnosis is sometimes necessary. For virus isolation, primary ovine or bovine cells, not routinely present, are needed. Serological methods exist, but electron microscopy is the most commonly used method. OBJECTIVES: To develop a reliable method for the laboratory diagnosis of orf zoonoses, without virus culture and without access to an electron microscope. STUDY DESIGN: A suitable primer pair was designed for orf polymerase chain reaction (PCR), using the Oligo software and sequence information from GenBank. Orf positive controls and specimens were kindly provided by several public health centers. Molluscum contagiosum specimens were provided by a dermatologist. HSV-1, HSV-2 and VZV positive swab specimens came from our routine diagnostic service. Asymptomatic skin specimens were obtained from sheep heads from the abattoir, and swab specimens from the heads of asymptomatic sheep. Selected amplified orf PCR positive specimens were sequenced to ensure the authenticity of the PCR products. Orf positive specimens were sent to another laboratory for electron microscopy. RESULTS AND CONCLUSIONS: A robust PCR was developed, with very small inter-run variation. All specificity demands were met, and the sensitivity seems to be good or excellent. All negative specificity controls from cell cultures and non-orf viruses were negative. Twenty-two (95.7%) of 23 scab or swab specimens with suspected orf etiology were orf PCR positive. Five of eight skin specimens from sheep heads from the abattoir were positive, and all 11 swab specimens from asymptomatic sheep were negative. Electron microscopy demonstrated orf-like particles in orf-PCR positive specimens. This PCR seems to be suitable as a diagnostic test for orf in humans, but asymptomatic virus shedding in sheep or goats may complicate veterinary applications of the assay.