Isolation of equine herpesvirus-5 from blood mononuclear cells of a gelding.

Horses are commonly infected by herpesviruses, but isolation of equine herpesvirus-5 (EHV-5) has only infrequently been reported. We describe the isolation and characterization of a strain of EHV-5 from the blood mononuclear cells of a healthy adult horse in California. The virus was initially identified by EHV-5 specific polymerase chain reaction (PCR), and it caused lytic infection of cultured rabbit kidney cells only after repeated serial passage. Virions with characteristic herpesvirus morphology were readily demonstrated in cell culture lysate by transmission electron microscopy. A portion of the glycoprotein B gene of this strain of EHV-5 had 99% identity to the published EHV-5 sequence, and it was clearly distinguishable from other EHV (1-4) by virus-specific PCR assays. Prevalence of EHV-5 infection in a group of young racehorses was estimated at 64% using the EHV-5 specific PCR on nasopharyngeal secretions.     

Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses.

In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys (Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging protein gene of EHV5 plus AsHV4, AsHV5, and a newly described putative pathogen herein designated asinine herpesvirus 6 (AsHV6). Phylogenetic analysis of these sequences suggested that the equine gammaherpesviruses may form a separate clade within the Gammaherpesvirinae subfamily. Based on the sequence of EHV2 and the novel sequences reported in this study, a PCR assay was developed to detect equine gammaherpesviruses. Products of the predicted size were produced after amplification of DNA from EHV2, EHV5, AsHV4, AsHV5, and AsHV6. This nonnested assay was shown to consistently amplify approximately 10 genomic copies of EHV2. Amplification products were not produced from DNA template of other alpha- and gammaherpesviruses. Because the role of gammaherpesviruses has not been well defined in equine disease, it is envisioned that a single, sensitive PCR assay to detect these potential pathogens will facilitate further assessment of their role in disease.     

Association of two newly recognized herpesviruses with interstitial pneumonia in donkeys (Equus asinus).

Over a period of 6 years, antemortem and postmortem examinations were performed on a number of donkeys suffering from respiratory disease. For many cases, initial diagnostic efforts failed to identify an etiology consistent with the pathologic findings. However, retrospective examination of these cases using consensus primer polymerase chain reaction, designed to recognize herpesviruses from all 3 subfamilies of the Herpesviridae, amplified a fragment of the highly conserved herpesvirus DNA polymerase gene from a number of these animals. Two novel herpesviruses, herein designated asinine herpesvirus 4 (AHV4) and asinine herpesvirus 5 (AHV5), were consistently detected in lung tissue from donkeys in which the histopathology was characterized by interstitial pneumonia and marked syncytial cell formation but not in lung tissue from donkeys with evidence of bacterial or verminous pneumonia. Nucleotide sequence and phylogenetic analysis places these new viruses within the Gammaherpesvirinae subfamily and indicates that they are most closely related to the recently identified zebra herpesvirus and wildass herpesvirus as well as equine herpesviruses 2 and 5.     

Equid herpesvirus 9 (EHV-9) isolates from zebras in Ontario,Canada, 1989 to 2007

The objective of this study was to identify and partially characterize 3 equid herpesviruses that were isolated postmortem from zebras in Ontario, Canada in 1989, 2002, and 2007. These 3 virus isolates were characterized by plaque morphology, restriction fragment length polymorphism (RFLP) of their genomic deoxyribonucleic acid (DNA), real-time polymerase chain reaction (PCR) assay, and sequence analyses of the full length of the glycoprotein G (gG) gene (ORF70) and a portion of the DNA polymerase gene (ORF30). The isolates were also compared to 3 reference strains of equid herpesvirus 1 (EHV-1). Using rabbit kidney cells, the plaques for the isolates from the zebras were found to be much larger in size than the EHV-1 reference strains. The RFLP patterns of the zebra viruses differed among each other and from those of the EHV-1 reference strains. Real-time PCR and sequence analysis of a portion of the DNA polymerase gene determined that the herpesvirus isolates from the zebras contained a G at nucleotide 2254 and a corresponding N at amino acid position 752, which suggested that they could be neuropathogenic EHV-1 strains. However, subsequent phylogenetic analysis of the gG gene suggested that they were EHV-9 and not EHV-1.     

Molecular diagnosis of alcelaphine herpesvirus (malignant catarrhal fever) infections by nested amplification of viral DNA in bovine blood buffy coat specimens

A fragment of alcelaphine herpesvirus-1 (AHV-1; malignant catarrhal fever) DNA was subcloned into pUC 18 and sequenced. The subclone hybridized strongly to AHV-1 DNA, weakly to alcelaphine herpesvirus-2 (AHV-2) DNA, and not at all to DNA from bovine herpesvirus-1 (BHV-1; infectious bovine rhinotracheitis [IBR] virus), bovine herpesvirus-2 (BHV-2; bovine herpes mamillitis [BHM] virus), and bovine herpesvirus-4 (BHV-4; isolate DN599). A 2-stage (nested) polymerase chain reaction (PCR) diagnostic test was devised based on a portion of the subcloned AHV-1 DNA sequence. First and second stage amplified AHV-1 DNA targets were 487 and 172 base pairs (bp) in length, respectively. Unique Pvu II and Stu I restriction endonuclease cleavage sites confirmed the identity of amplified AHV-1 DNA. Five AHV-1 and 2 AHV-2 isolates were identically and specifically PCR positive. BHV-1, BHV-2, and BHV-4 viruses were negative by the same procedure. As little as 0.01 TCID50 AHV-1 was detected using the nested amplification procedure. Simple methods of buffy coat isolation from bovine blood were employed to prepare specimens for PCR. An AHV-1-infected calf was PCR positive from 3 to 77 days postinoculation (PI), with rising seroconversion first noted 14 days PI. The AHV-1 DNA sequence was 62% homologous to a portion of the Epstein-Barr virus genome. The nested PCR procedure may improve the viral diagnosis of clinical and subclinical alcelaphine herpesvirus infections.     

Prevalence of equine herpesvirus-1 and equine herpesvirus-4 infections in equidae species in Turkey as determined by ELISA and multiplex nested PCR

In this report we examined the presence of specific antibodies against equine herpesvirus type 1 (EHV-1), and equine herpesvirus type 4 (EHV-4) in several equidae, including mules, donkeys, horses. The presence of EHV-1 and EHV-4 in respiratory diseases of equids, and ability of multiplex nested polymerase chain reaction (PCR) screening in simultaneous diagnosis of horses acutely infected by EHV-1 and EHV-4 were also investigated. Sera from 504 horses, mules and donkeys sampled were tested for the presence of EHV-1 and EHV-4 specific antibodies. Blood samples taken from 21 symptomatic horses and nasal swabs taken from 40 symptomatic horses were tested for the presence of EHV-1 and EHV-4 by a multiplex nested PCR. A total of 14.3% (3/21) of buffy coat samples and 32.5% (13/40) nasal swab samples were found to contain EHV-1 DNA, while 19% (4/21) buffy coat samples and 22.5% (9/40) nasal swab samples were found to be positive for EHV-4 DNA. By species, 14.5% of horses, 37.2% of mules and 24.2% of donkeys tested were EHV-1 seropositive. EHV-4 specific antibodies were detected in 237 (81.7%) of 290 horse sera tested. Results from this investigation demonstrate that EHV-1 and EHV-4 are prevalent throughout the equid population, and that donkeys and mules might also represent an important source of infection for other equids. We also showed that the multiplex nested PCR assay might be useful for diagnosis of mixed respiratory infections in horses due to EHV-1 and EHV-4.     

Molecular phylogenetic analysis of felid herpesvirus 1.

The position of felid herpesvirus 1 within the alphaherpesvirus subfamily was investigated using molecular phylogenetic techniques applied to multiple sequence alignments of recently reported FHV-1 gene homologs (glycoprotein B, ribonucleotide reductase and DNA polymerase). FHV-1 was most closely related to other carnivore alphaherpesviruses, (phocid herpesvirus 1 and canid herpesvirus 1) and to the equid herpesviruses 1 and 4.     

Lack of Detectable Equine Herpesviruses 1 and 2 in Paraffin-Embedded Specimens of Equine Sarcoidosis

Background: Equine sarcoidosis is a rare, multisystemic, noncaseating, granulomatous and lymphoplasmacytic disease of unknown etiology. A recent report described a horse with granulomatous skin disease displaying histologic, electron microscopic, and polymerase chain reaction (PCR) findings consistent with equine herpesvirus 2 (EHV-2).
Objective: To investigate the presence of EHV-2 and equine herpesvirus 1 (EHV-1) in 8 horses with sarcoidosis.
Animals: Eight horses with sarcoidosis, reported previously.
Methods: Retrospective study. PCR assays of the tissues were performed to detect DNA associated with EHV-1 and EHV-2. For both herpesviruses the target was their respective glycoprotein B gene. Positive controls consisted of DNA from viral cultures of culturettes from naturally occurring respiratory infections of EHV-1 and EHV-2.
Results: The PCR analyses for both equine herpesviruses' DNA were negative in all 8 horses.
Conclusion: The failure to detect DNA from EHV-1 and EHV-2 in paraffin-embedded skin of these 8 horses does not discount EHV-1 or EHV-2 as causing some cases of ES, but lends support to the presumably multifactorial etiologic nature of the disease.     

Prevalence of antibodies to alcelaphine herpesvirus-1 and nucleic acid hybridization analysis of viruses isolated from captive exotic ruminants

A serologic survey was conducted to determine the prevalence of antibodies to alcelaphine herpesvirus-1 (AHV-1) in captive exotic ruminants within the United States. Forty-six percent of the members of the subfamily Alcelaphinae (wildebeest, topi, hartebeest) in the family Bovidae had virus-neutralizing antibody to AHV-1. Other subfamilies of Bovidae with high prevalence of virus-neutralizing antibodies to AHV-1 included Hippotraginae (oryx and addax) and Caprinae (sheep and goats), with prevalence of 45% and 29%, respectively. Herpesviruses that have been isolated from captive exotic ruminant species, including healthy animals and those with clinical malignant catarrhal fever at the Oklahoma City Zoo and the San Diego Zoo/Wild Animal Park, were analyzed by DNA restriction enzyme analysis and blot hybridization. Variation has been detected among the genomes of several malignant catarrhal fever virus isolates obtained from various exotic species of ruminants, using the DNA restriction enzymes BamHI and HindIII. The DNA of these virus isolates is distinct from that of bovine herpesviruses 1, 2, and 4, as demonstrated by restriction enzyme analysis and nucleic acid hybridization. On the basis of restriction enzyme analysis and nucleic acid hybridization data, the DNA from each of the putative alcelaphine herpesvirus isolates examined, except for the topi virus isolate, had a high degree of DNA sequence similarity with the original AHV-1 isolate, WC-11, from a blue wildebeest.     

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Identification of a novel herpesvirus from a California desert tortoise (Gopherus agassizii)

Herpesviruses are significant pathogens of tortoises, causing upper respiratory tract disease and necrotizing stomatitis, with infections often associated with high mortality rates. Herpesvirus infection in a captive California desert tortoise (Gopherus agassizii) was detected by light microscopic observation of intranuclear inclusion bodies in various tissues followed by transmission electron microscopic observation of herpesvirus-like particles, and amplification of herpesvirus nucleic acid sequences using polymerase chain reaction. Using an indirect enzyme linked immunosorbent assay, anti-tortoise herpesvirus antibodies were detected one month after initial onset of clinical signs. This novel herpesvirus is distinct from the previously described tortoise herpesvirus (tortoise herpesvirus-1, THV-1) sharing 83% sequence identity of 60 amino acids of a portion of the DNA polymerase gene and 79% sequence identity across 120 amino acids of a portion of the ribonucleotide reductase gene. Similar to THV-1, this novel herpesvirus, tortoise herpesvirus-2 (THV-2), also clusters with the alphaherpesviruses.     

Equine Multinodular Pulmonary Fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: a case report

A standardbred gelding with a history of 10 days pyrexia and lethargy was referred to the Equine Hospital at the Swedish University of Agricultural Sciences in Uppsala, Sweden.The horse had tachypnea with increased respiratory effort and was in thin body condition. Laboratory findings included leukocytosis, hyperfibrinogenemia and hypoxemia. Thoracic radiographs showed signs of pneumonia with a multifocal nodular pattern, which in combination with lung biopsy findings indicated Equine Multinodular Pulmonary Fibrosis (EMPF). EMPF is a recently described disease in adult horses with clinical signs of fever, weight loss and respiratory problems. The pathological findings include loss of functional pulmonary parenchyma due to extensive nodular interstitial fibrosis which has been related to infection with the equine herpesvirus type 5 (EHV-5). In this case, lung biopsy and tracheal wash samples tested positive for both asinine herpesvirus type 5 (AHV-5) and EHV-5 using PCR assays. The horse failed to respond to treatment and was euthanized for humane reasons. Postmortem examination confirmed the diagnosis of EMPF. This case suggests that not only EHV-5 alone should be considered in association with the development of this disease.     

Isolation of equine herpesvirus-2 from the lung of an aborted fetus.

This study describes the isolation of equine herpesvirus-2 (EHV-2) from the lung of an aborted equine fetus in Argentina. The isolated virus was confirmed as EHV-2 by indirect immunofluorescence using a rabbit anti-EHV-2 polyclonal antiserum and by virus-neutralization test using an equine polyclonal antibody against EHV-2. Restriction endonuclease DNA fingerprinting with BamHI also confirmed the identity of the virus as EHV-2. Furthermore, viral nucleic acid was detected by polymerase chain reaction from the original lung sample and from the DNA obtained from cells infected with the virus isolate. This work constitutes the first reported isolation of EHV-2 from an aborted equine fetus. The presence of EHV-2 in the lung of the aborted fetus would indicate that this virus is capable of crossing the placental barrier. However, no cause-effect relationship was established between the EHV-2 isolate and the abortion.     

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.     

Revised phylogenetic relationships among herpesviruses isolated from sturgeons

Initial phylogenetic comparisons based on a region of the DNA polymerase of seven herpes-like viruses found in sturgeons in North America and Europe indicated the presence of three distinct clades. A revised phylogenetic analysis of the same viruses, based on corrected DNA polymerase sequences and newly obtained sequence data from the putative ATP subunit of the terminase gene, indicate only two clades. These two clades correspond to the historical designations given to these herpes-like viruses from white sturgeon Acipenser transmontanus: white sturgeon herpesvirus type 1 (WSHV-1) and type 2 (WSHV-2). The identification of putative terminase gene sequences for all seven herpes-like viruses from sturgeons confirms their affinity with the family Herpesviridae (because this gene is unique to herpesviruses) and more distantly with T4-like bacteriophages. The two clades of sturgeon herpesviruses are therefore appropriately designated as Acipenserid herpesviruses 1 and 2, which correspond to the previous common names of white sturgeon herpesvirus types 1 and 2.     

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 113 bp 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.     

Temporal detection of equine herpesvirus infections of a cohort of mares and their foals

The objectives of this study were to estimate the prevalence of equine herpesviruses (EHV) 1-5 in the nasal secretions (NS) of a cohort of 12 mares and their foals from birth to 6 months of age, estimate the prevalence of EHV-1-5 infection of peripheral blood mononuclear cells (PBMC) of selected foals, and investigate phylogenetic relationships amongst the various strains of EHV-2 and 5. Virus-specific PCR assays were used to detect EHV-1-5 in NS and PBMC. A homologous portion of the glycoprotein B (gB) gene of the various strains of EHV-2 and 5 was sequenced and compared. EHV-2, 4, and 5 were all detected in NS from the horses, but only EHV-4 was associated with respiratory disease (P=0.005). EHV-2 and 5 infections were both common, but foals shed EHV-2 in their NS earlier in life than EHV-5 (P=0.01). Latent EHV-2 and 5 infections were detected in the PBMC of 75 and 88%, respectively, of the foals at approximately 6 months of age. The strains of EHV-2 shed in the NS of individual horses were more genetically heterogeneous than the strains of EHV-5 (95.5-99.3% versus 98.8-99.3% nucleotide identity, respectively). One-month-old foals typically shed strains of EHV-2 that were identical to those infecting their dams whereas older foals often shed virus strains that were different from those of their dams. Although herpesvirus infections were ubiquitous in this cohort of horses, there were distinct clinical consequences and clear epidemiological differences between infections with the different viruses.     

Derivation of a DNA clone corresponding to the viral agent of sheep-associated malignant catarrhal fever

Malignant catarrhal fever is a fatal lymphoproliferative and degenerative disease of ruminants. One causative agent is the gammaherpesvirus alcelaphine herpesvirus 1 (AHV-1), which produces no disease in its natural host, the wildebeest (Connochaetes species). Epidemiological evidence implicates sheep as the carrier of a similar virus. However, attempts to culture this virus from sheep or from animals affected with sheep-associated malignant catarrhal fever (SA-MCF) have failed. Lymphoblastoid cells have been propagated from cattle, deer and rabbits with SA-MCF. Although these cells show no evidence of viral particles or antigens, hybridisation experiments now show that they contain DNA sequences homologous to those of AHV-1. A genomic library was constructed from one of these lymphoblastoid cell lines and a clone identified which hybridised to cloned AHV-1 DNA. The authors believe that this clone contains part of the SA-MCF viral genome, and that the SA-MCF virus and AHV-1 are closely related gammaherpesviruses.     

Isolation and characterisation of cervine herpesvirus-1 from red deer semen

AIM: This communication describes the isolation of herpesvirus during routine export examination of semen collected from red deer stags in New Zealand. METHODS: Virus isolation was carried out using bovine embryonic lung (BEL) cells and viruses were characterised by direct immunofluorescense, restriction-fragment-length polymorphism analysis (RFLP), polymerase chain reaction (PCR) analysis and nucleotide sequencing. RESULTS: Herpesvirus was isolated from red deer semen on 2 different occasions from different animals. In both cases the virus was identified as cervine herpesvirus-1 (CvHV-1), based on RFLP, PCR and sequence analysis. Nucleotide sequence analysis of the glycoprotein-D gene showed 99.7% homology to the Banffshire strain of CvHV-1 and 89.5%, 89.2%, 85.3% and 79.6% homology to bovine herpesvirus 1.2 (BoHV-1.2), bovine herpesvirus 1.1 (BoHV-1.1), cervine herpesvirus-2 (CvHV-2) and caprine herpesvirus-1 (CpHV-1), respectively. CONCLUSION: This is the first time that CvHV-1 has been isolated in New Zealand. Its inclusion in serological surveys will allow the prevalence of CvHV-1 in the red deer population to be assessed in this country. The clinical significance of CvHV1 infection in New Zealand red deer herds has yet to be determined.