Cervid Herpesvirus Research Articles

Experimental Infection of Reindeer with Cervid Herpesvirus 2

Cervid herpesvirus 2 (CvHV2) has been isolated from reindeer (Rangifer tarandus tarandus), and serological data indicate that in reindeer this virus is endemic in Fennoscandia, Alaska, Canada, and Greenland. CvHV2 has been described as a cause of subclinical genital infections in reindeer, but little information on primary infections exists. In this study, six seronegative and presumably pregnant reindeer were allocated to one of two groups. Two animals were inoculated with CvHV2 intratracheally, and two animals intravaginally, with one control animal in each group receiving sterile water. Mild hyperthermia and serous discharges from the vagina and nose were observed. No abortions were recorded, but one calf died shortly after birth. Inoculated animals seroconverted and had neutralizing antibodies after days 7 to 10 postinfection. CvHV2 was detected by PCR in nasal and vaginal swabs from animals in both groups but could be isolated only from nasal swabs in the respiratory group and from vaginal swabs in the genital group. CvHV2 was detected by PCR in various organs and tissues postmortem. In control animals, the virus could not be isolated in spite of PCR-positive nasal and vaginal swab samples and some degree of positive immunostaining. One of the animals that were inoculated intratracheally developed a hemorrhagic, necrotizing bronchopneumonia, which was CvHV2 positive by PCR and immunohistochemistry. We conclude that CvHV2 can cause systemic infection, that both genital and respiratory inoculations can lead to virus shedding, and that the virus can infect the fetus in utero.

Cervid Herpesvirus 2, the Primary Agent in an Outbreak of Infectious Keratoconjunctivitis in Semidomesticated Reindeer

An outbreak of infectious keratoconjunctivitis (IKC) occurred in semidomesticated reindeer (Rangifer tarandus tarandus) in Troms County, Norway, in February 2009. Twenty-eight animals with clinical symptoms and 12 apparently healthy animals were investigated. They ranged in age from calves of the year to 4-year-old animals (mean, 1.9 years; standard deviation, +/-0.9). The seroprevalence of antibodies against cervid herpesvirus 2 (CvHV2) was 86% in animals with IKC and 42% in unaffected animals. For the 28 clinically affected animals, CvHV2 was detected by PCR in swabs obtained from the eye (82%), nose (64%), and vagina (24%), and CvHV2 was isolated from eye swabs from 8 animals. Virus was not isolated from clinically unaffected animals but was detected by PCR in eye swab samples from five of them. The viral activity, assessed by the ability to cause a cytopathic effect in cell culture, increased with the severity of clinical symptoms, but in severe clinical cases, virus was absent and secondary bacterial infections were dominant. Moraxella sp. isolates were obtained from seven animals, and those from two animals were identified as Moraxella bovoculi. Staphylococcus aureus, Streptococcus sp., and Arcanobacterium pyogenes were also isolated. It is concluded that CvHV2, which is endemic in reindeer, can cause IKC, probably most commonly as a primary infection of calves. This can be a very painful and devastating disease of economic importance for reindeer herders. This is the first report of CvHV2 as the primary agent of IKC in reindeer. This is also the first isolation of this virus in reindeer under natural herding conditions.

Cervid herpesvirus 2 experimentally reactivated in reindeer can produce generalized viremia and abortion

Cervid herpesvirus 2 (CvHV2) has never been isolated from reindeer in Norway, but serological data and investigations by PCR indicate that the virus is endemic in the country, with horizontal and vertical transmission, systemic spread, and latency in the trigeminal ganglion. In this study two seropositive reindeer, one of which was pregnant, were administered dexamethasone, to reactivate CvHV2 latent infection. One control animal received sterile water. All animals including the control reactivated, as shown by amplification of CvHV2 DNA from nasal swabs. The pregnant animal showed lesions in the lip mucosa 10 days after the first dexamethasone injection and CvHV2 was visualized by electron microscopy and isolated from those lesions, as well as from nasal and vaginal swabs. On day 13 she aborted and CvHV2 was isolated from both the aborted calf and the mother. CvHV2 was isolated from the other animal administered dexamethasone. Despite amplification of viral DNA in the control animal, it was never possible to isolate the virus. Molecular characterization of the new isolates confirmed these to be CvHV2, and similar to the previous known strain Salla82. Present results represent the first isolation of CvHV2 in Norway and reconfirm that this virus can cause systemic infections in reindeer even after reactivation episodes, and infect the fetus in utero despite a prompt immune response. While it is not possible to atribute the abortion to CvHV2 alone, present data together with previous reports of vertical transmission of CvHV2 and neonatal death, point to an abortogenic potential, which should be further investigated.

Alphaherpesvirus infections in semidomesticated reindeer: A cross-sectional serological study

Alphaherpesviruses infect a wide range of animal species and cause diseases. Cervid herpesvirus 2 (CvHV-2) was originally isolated from reindeer in Finland but the impact of CvHV-2 infections on reindeer remains unclear. CvHV-2 infection could be partly responsible for calf losses as there are indications that it is associated with abortions and neonatal diseases. Previous serosurveys of reindeer (Rangifer tarandus tarandus) have shown that an alphaherpesvirus is circulating among reindeer in Norway. The aim of the present study was to determine the prevalence of CvHV-2 infection among reindeer in various herding districts in Finnmark, the largest reindeer area in Norway, and to identify factors associated with becoming infected with CvHV-2. A total of 3062 serum samples were tested using an ELISA and a sub-set of samples was further tested using a seroneutralization test. The ELISA revealed that 49% of samples were positive. Extrapolation of the results to the total population (111,350 animals; 66% of the Finnmark reindeer population) showed that the seroprevalence in the population was 48%. Seroprevalence varied from 7.6% to 90.7% between districts and was affected by age, weight and population density. ELISA-positive samples neutralized CvHV-2 at serum dilutions greater than those required for neutralization of bovine herpesvirus type 1. It is concluded that CvHV-2 is endemic throughout the reindeer herding districts of northern Norway.

Cervid herpesvirus 2 causes respiratory and fetal infections in semidomesticated reindeer.

Members of the viral subfamily Alphaherpesvirinae establish latency from which they can be reactivated. Bovine herpesvirus 1 causes infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cattle, as well as abortion and weak calves. Serological evidence of alphaherpesvirus infection has been reported for wild and semidomesticated reindeer (Rangifer tarandus tarandus) in Norway. To address the possibility that reindeer alphaherpesvirus (cervid herpesvirus 2 [CvHV-2]) infection might affect the respiratory system and in part explain the relatively high mortality of reindeer calves during their first year, tissue samples were obtained from reindeer and reindeer fetuses at slaughterhouses in Finnmark County, Norway. A nested pan-alphaherpesvirus PCR amplification targeting the highly conserved UL27 gene (encoding glycoprotein B) was used. Sequencing of amplicons revealed the presence of CvHV-2 DNA. The detection of CvHV-2 DNA in trigeminal ganglia (27 of 143 samples), nasal swabs (5 of 75 samples), and fetal tissues (12 of 48 samples) indicates that CvHV-2 infection is endemic in this reindeer population. Moreover, the virus is transmitted horizontally by the respiratory route, establishing latency in the trigeminal ganglion, and vertically to the fetus through the placenta. Further studies should focus on the reproductive impact of CvHV-2 infection in reindeer.

Evaluation of three commercial bovine ELISA kits for detection of antibodies against Alphaherpesviruses in reindeer (Rangifer tarandus tarandus)

BackgroundThe genus Varicellovirus (family Herpesviridae subfamily Alphaherpesvirinae) includes a group of viruses genetically and antigenically related to bovine herpesvirus 1 (BoHV-1) among which cervid herpesvirus 2 (CvHV-2) can be of importance in reindeer. These viruses are known to be responsible for different diseases in both wild and domestic animals. Reindeer are a keystone in the indigenous Saami culture and previous studies have reported the presence of antibodies against alphaherpesviruses in semi-domesticated reindeer in northern Norway. Mortality rates, especially in calves, can be very high in some herds and the abortion potential of alphaherpesvirus in reindeer, unlike in bovines, remains unknown.ELISA kits are the most used screening method in domestic ruminants and given the close genetic relationship between viruses within this genus, it might be possible to use such kits to screen cervids for different alphaherpesviruses. We have compared three different commercial ELISA kits in order to validate its use for reindeer and CvHV-2.MethodsThree commercial bovine ELISA kits (A, B and C), using either indirect (A) or blocking (B and C) ELISA techniques to detect antibodies against BoHV-1 were tested with sera from 154 reindeer in order to detect antibodies against CvHV-2. A Spearman's rank-based coefficient of correlation (ρ) was calculated. A dilution trial was performed for all kits. A virus neutralization test using both BoHV-1 and CvHV-2 was carried out.ResultsSeroprevalence was almost the same with all kits (40–41%). Despite a similar qualitative score, quantitatively kits classified samples differently and a strong correlation was only identified between Kits B and C. Blocking kits performed better in both repeatability and in the dilution trial. The virus neutralization results confirmed the ELISA results to a very high degree. Neutralizing titres ranged from 1:2 to 1:256 and from 0 to 1:16 against CvHV-2 and BoHV-1 respectively.ConclusionResults show that the genetic and antigenic similarity between BoHV-1 and CvHV-2 enables the use of a bovine gB blocking ELISA kit to screen reindeer. The use of an ELISA kit is both cheaper and time saving, allowing screening of large populations. This study revealed a high number of positive animals against CvHV-2 and its impact and distribution in the general population should be further evaluated.

Isolation and characterisation of a ruminant alphaherpesvirus closely related to bovine herpesvirus 1 in a free-ranging red deer.

BackgroundThe genus Varicellovirus of the Herpesviridae subfamily Alphaherpesvirinae includes a cluster of viruses antigenically and genetically related to bovine herpesvirus 1 (BoHV-1): namely bovine herpesvirus 5 (BoHV-5), bubaline herpesvirus 1 (BuHV-1), caprine herpesvirus 1 (CpHV-1), cervid herpesviruses 1 (CvHV-1) and 2 (CvHV-2) and elk herpesvirus 1 (ElkHV-1). Considering the serological relationship between these ruminant alphaherpesviruses, several surveys have studied the occurrence of BoHV-1 related virus infection in wild and domestic ruminant species. In this way, a recent investigation has indicated, in Belgium, a high increase in the serological prevalence of BoHV-1 related virus infection in free-ranging red deer population. In this context, it has been decided to investigate the presence of an alphaherpesvirus spreading in the Belgian free-ranging red deer population.ResultsThe current study reports the first isolation in a free-ranging red deer of a BoHV-1 closely related virus. The isolate was antigenically, genomically and genetically characterised by comparison with several ruminant alphaherpesvirus. Immunofluorescence assays revealed the isolate was antigenically distinct from bovine and caprine alphaherpesviruses. Similarly, BamHI and BstEII restriction analyses demonstrated the genomic difference between the isolate and the other ruminant alphaherpesviruses. Next, the sequencing of selected parts of UL27 and US8 genes showed a high degree of homologies between each BoHV-1 related ruminant alphaherpesvirus and the isolate. Besides the close relationship between all ruminant alphaherpesviruses, the phylogenetic analysis revealed that the isolate clustered with CvHV-1.ConclusionThe first isolation of a virus closely related to BoHV-1 in a free-ranging red deer is reported. Data demonstrate that a CvHV-1 strain, named Anlier, circulates in wild red deer in continental Europe. Anlier strain show consistent differences with the virus isolated from Scottish farmed red deer. All together, these results improve our understanding of ruminant alphaherpesviruses.

Serological Survey for Potential Disease Agents of Free-ranging Cervids in Six Selected National Parks from Germany

A total of 164 blood samples, collected from free-ranging red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in six German national parks (NP) between 2000 and 2002, were assayed for antibodies against nine viral disease agents. Antibodies were only detected against the alpha-herpesviruses; specifically, bovine herpesvirus-1 (BHV-1) (22 of 157, 14%), cervid herpesvirus-1 (17 of 157, 10.8%), and caprine herpesvirus-1 (11 of 159, 6.9%). Titers ranged from 4 to 102. Most of the seropositive sera, and those with the highest antibody titers, were from red and roe deer in the Harz and Hochharz NP, which are connected and allow migration between the two. The distribution and specificity of antibodies detected in individual deer suggests that the three alpha-herpesviruses are circulating in these deer populations. No antibodies were detected against bovine viral diarrhea virus, epizootic hemorrhagic disease virus, bovine leukemia virus, bluetongue virus, foot-and-mouth disease virus, or sheep and goat poxvirus.

Ruminant alphaherpesviruses related to bovine herpesvirus 1

Herpesviruses have mainly co-evolved with their hosts for millions of years. Consequently, different related host species may have been infected by various genetically related herpesviruses. Illustrating this concept, several ruminant alphaherpesviruses have been shown to form a cluster of viruses closely related to bovine herpesvirus 1 (BoHV-1): namely bovine herpesvirus 5, bubaline herpesvirus 1, caprine herpesvirus 1, cervid herpesviruses 1 and 2 and elk herpesvirus 1. These viruses share common antigenic properties and the serological relationships between them can be considered as a threat to BoHV-1 eradication programmes. BoHV-1 is a herpesvirus responsible for infectious bovine rhinotracheitis, which is a disease of major economic concern. In this article, the genetic properties of these ruminant alphaherpesviruses are reviewed on a comparative basis and the issue of interspecific recombination is assessed. The pathogenesis of these infections is described with emphasis on the host range and crossing of the host species barrier. Indeed, the non bovine ruminant species susceptible to these ruminant alphaherpesviruses may be potential BoHV-1 reservoirs. The differential diagnosis of these related infections is also discussed. In addition, available epidemiological data are used to assess the potential of cross-infection in ruminant populations. A better knowledge of these ruminant alphaherpesvirus infections is essential to successfully control infectious bovine rhinotracheitis.

Phylogeny and antigenic relationships of three cervid herpesviruses

Elk herpesvirus (ElkHV) from North American elk (wapiti, Cervus elaphus nelsoni) is a recently identified alphaherpesvirus related to bovine herpesvirus-1 (BHV-1). In this study, we determined its relationship with European cervid herpesviruses: cervid herpesvirus-1 (CerHV-1) from red deer and rangiferine herpesvirus (RanHV) from reindeer. For phylogenetic analysis, genes for the gC and gD proteins of these viruses were sequenced. These genes demonstrated an extremely high GC content (76–79%). Genetically, ElkHV was found to be closely related to CerHV-1 and both viruses are more closely related to BHV-1 than to RanHV. Antigenically, the same relationships were found. ElkHV shares common neutralizing epitopes with both CerHV-1 and RanHV. A total of 10 epitopes were defined on the gB, gC and gD proteins of these viruses, including a shared neutralizing epitope on gD. The results indicate that ElkHV and CerHV-1 have diverged from a common ancestor virus. Cervid herpesviruses may be useful in determination of evolutionary rates of change for alphaherpesvirus genes.

Initial experiences with consensus primer PCR for detection of animal herpesviruses

Consensus primer polymerase chain reaction (CP-PCR) is a useful technique for the detection and partial characterisation of novel viruses of veterinary importance. CP-PCR differs from conventional PCR, in that the upstream and downstream primers each consist of a mixture of slightly different oligonucleotides, rather than a single, defined sequence. As used in virus discovery, CP-PCR amplifies a segment of the genome of all members of a particular virus group. Conserved virus genes, e.g. those encoding enzymes, are the most suitable targets when designing these strategies. We used a previously validated CP-PCR method to amplify herpesviral DNA from the left conjunctival sac of a farmed red deer (Cervus elaphus) with keratitis. Sequencing of the CP-PCR product revealed 97% homology to a 174-base pair segment of a novel rhadinovirus of elk (Alces alces) (GenBank AY237365.1). An attempt to isolate a herpesvirus from the deer's conjunctival sac in tissue culture was unsuccessful, as was PCR using conventional primers specific for Cervid herpesvirus 1. This case exemplifies the potential value of CP-PCR for detection of novel herpesviruses.

SEROLOGIC SURVEILLANCE FOR SELECTED VIRAL AGENTS IN CAPTIVE AND FREE-RANGING POPULATIONS OF ARABIAN ORYX (ORYX LEUCORYX) FROM SAUDI ARABIA AND THE UNITED ARAB EMIRATES

A total of 294 sera collected between 1999 and 2001 from eight captive and one free-ranging herds of Arabian oryx (Oryx leucoryx) distributed in Saudi Arabia (SA) and the United Arab Emirates (UAE) were assayed for antibodies against 13 selected viral agents. Arabian oryx have been exposed to bluetongue virus (BTV), epizootic hemorrhagic disease virus (EHDV), rinderpest virus (RPV), bovine respiratory syncytial virus (BRSV), bovine adenovirus 3 (BAV-3), cervid herpesvirus-1, foot-and-mouth disease virus, equine herpesvirus 9, and bovine viral diarrhea virus. The high seroprevalence to BTV and EHDV in the UAE and SA indicates that Arabian oryx are likely to be susceptible to infection by these viruses and therefore could act as a source of virus to vectors during the infective stage of infection. Moreover, antibodies were detected against RPV and BRSV in sera from SA and against BAV-3 in sera from the UAE. No antibodies were found against bovine herpesvirus-1, caprine herpesvirus-1, enzootic bovine leucosis virus, and peste des petits ruminants virus. On the basis of these results, caution should be applied when considering translocation of Arabian oryx, and only those proven to be free of infectious agents that might present a risk to other species should be moved.

ANTIBODIES TO RUMINANT ALPHA-HERPESVIRUSES AND PESTIVIRUSES IN NORWEGIAN CERVIDS

A serologic survey revealed that Norwegian populations of free-ranging reindeer (Rangifer tarandus tarandus), roe deer (Capreolus capreolus), red deer (Cervus elaphus), and moose (Alces alces) have been exposed to alpha-herpesviruses and pestiviruses. A total of 3,796 serum samples collected during the period 1993-2000 were tested in a neutralization test for antibodies against bovine herpesvirus 1 (BHV-1) or cervid herpesvirus 2 (CerHV-2), and 3,897 samples were tested by a neutralization test and/or enzyme-linked immunosorbent assay for antibodies against bovine viral diarrhea virus (BVDV). Antibodies against alpha-herpesvirus were found in 28.5% of reindeer, 3.0% of roe deer, and 0.5% of red deer, while all moose samples were negative. In reindeer, the prevalence of seropositive animals increased with age and was higher in males than females. Antibodies against BVDV were detected in 12.3% of roe deer, 4.2% of reindeer, 2.0% of moose and 1.1% of red deer. The results indicate that both alpha-herpesvirus and pestivirus are endemic in reindeer and pestivirus is endemic in roe deer in Norway. The viruses may be specific cervid strains. Seropositive red deer and moose may have become exposed as a result of contact with other ruminant species.

Virologic investigations of free-living European bison (Bison bonasus) from the Bialowieza Primeval Forest, Poland.

We conducted virologic investigations on postmortem specimens from 261 free-living European bison (Bison bonasus) from the Bialowieza Primeval Forest, Poland collected between 1990 and 2000. Fifty-four of 94 males had balanoposthitis; none of the 167 female bison examined had reproductive tract lesions. Peripheral blood, swabs, and various tissues were analyzed for bovine viruses as well as for viral DNA by bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 4 (BoHV-4) specific polymerase chain reaction (PCR) technique. An infectious bovine rhinotracheitis like BoHV-1 strain was isolated from the spleen of a female bison calf and additionally was detected by nested PCR from splenic tissue. None of the bison had significant antibody titers against BoHV-1, bovine herpesvirus 2, BoHV-4, caprine herpesvirus 1, cervid herpesvirus 1, or bovine viral diarrhea (BVD) virus-1. However, low antibody titers in two animals indicate that this European bison population has been exposed to BVD virus or BVD-like viruses and BoHV-2.

Keratoconjunctivitis in a free-ranging red deer(Cervus elaphus) population in Spain

This study describes the occurrence of clinical keratoconjunctivitis in a Spanish red deer(Cervus elaphus) population 150 km south of Madrid. Necropsy findings, microbiological and serological results are reported. In September 1996 a total of 28 carcasses of stags were inspected, of which 11 (39%) showed macroscopic lesions of keratitis or keratoconjunctivitis. Conjunctival swabs and frozen tissue samples of the eyeballs were taken from six stags and used for isolation trials ofMoraxella sp. andMycoplasma sp., Smears and histological sections were stained with Stamp technique for the detection ofChlamydia psittaci. None of these pathogenic organisms could be detected. Serum samples were taken from 19 red deer of different age and sex and tested for antibodies againstChlamydia psittaci by enzyme linked immunosorbent assay as well as against three different alpha-herpesviruses (bovine herpesvirus-1, caprine herpesvirus-1, cervid herpesvirus-1) by virus neutralization test. Antibodies againstChlamydia psittaci were detected in 16 samples (84%), while none of the tested individuals was seropositive against any of the three alpha-herpesviruses. The serological results indicate, that this Spanish red deer population has been exposed toChlamydia psittaci and therefore this pathogen agent might be involved in the infection process of keratoconjunctivitis in this deer population.

Restriction maps of the DNA of cervid herpesvirus 1 and cervid herpesvirus 2, two viruses related to bovine herpesvirus 1.

Restriction maps of cervid herpesviruses 1 and 2 which are antigenetically related to bovine herpesvirus 1, were deduced from Southern blot hybridization with HindIII restriction fragments of BHV-1 DNA as probes.

Latent Herpesvirus Infection in Red Deer: Characterization of a Specific Deer Herpervirus Including Comparison of Genomic Restriction Fragment Patterns

Glucocorticoid treatment of imported red deer (Cervus elaphus), seropositive to Infectious Bovine Rhinotracheitis (IBR) virus, reactivated a latent herpesvirus infection, which was transmitted to a seronegative deer with a fatal outcome. However the virus did not spread to cattle housed in close contact with the infected deer, and serological indication og infection in the cattle was observed only on direct nasal installation of virus. The virus isolate had characteristics in common with other Alpha herpesviruses and especially the Bovid Herpesvirus type 1 (BHV-1) but distinguished itself from the latter by its host specificity, serological reaction and genomic restriction fragment pattern (RFP). The host specific red deer herpesvirus was tentatively designated Cervid Herpesvirus type 1 (CHV-1). It was concluded that CHV-1 seropositive deer can be a threat to red deer farming, while in cattle the infection may only cause minor inconvenience through interference with the serological IBR diagnosis.