Epidemiological and Molecular Analysis of an Outbreak of Highly Pathogenic Avian Influenza H5N8 clade 2.3.4.4 in a German Zoo: Effective Disease Control with Minimal Culling.

Outbreaks of highly pathogenic avian influenza A virus (HPAIV) subtype H5N8, clade 2.3.4.4, were first reported in January 2014 from South Korea. These viruses spread rapidly to Europe and the North American continent during autumn 2014 and caused, in Germany, five outbreaks in poultry holdings until February 2015. In addition, birds kept in a zoo in north-eastern Germany were affected. Only a few individual white storks (Ciconia ciconia) showed clinical symptoms and eventually died in the course of the infection, although subsequent in-depth diagnostic investigations showed that other birds kept in the same compound of the white storks were acutely positive for or had undergone asymptomatic infection with HPAIV H5N8. An exception from culling all of the 500 remaining zoo birds was granted by the competent authority. Restriction measures included grouping the zoo birds into eight epidemiological units in which 60 birds of each unit tested repeatedly negative for H5N8. Epidemiological and phylogenetical investigations revealed that the most likely source of introduction was direct or indirect contact with infected wild birds as the white storks had access to a small pond frequented by wild mallards and other aquatic wild birds during a period of 10 days in December 2014. Median network analysis showed that the zoo bird viruses segregated into a distinct cluster of clade 2.3.4.4 with closest ties to H5N8 isolates obtained from mute swans (Cygnus olor) in Sweden in April 2015. This case demonstrates that alternatives to culling exist to rescue valuable avifaunistic collections after incursions of HPAIV.

Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.

It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years.

Emergence of a novel cluster of influenza A(H5N1) virus clade 2.2.1.2 with putative human health impact in Egypt, 2014/15.

A distinct cluster of highly pathogenic avian influenzaviruses of subtype A(H5N1) has been found to emergewithin clade 2.2.1.2 in poultry in Egypt since summer2014 and appears to have quickly become predominant.Viruses of this cluster may be associated withincreased incidence of human influenza A(H5N1) infectionsin Egypt over the last months.

Nucleic acid-based detection of influenza A virus subtypes H7 and N9 with a special emphasis on the avian H7N9 virus.

In 2013, a novel influenza A virus of subtype H7N9 was transmitted from avian sources to humans in China, causing severe illness and substantial mortality. Rapid and sensitive diagnostic approaches are the basis of epidemiological studies and of utmost importance for the detection of infected humans and animals. We developed various quantitative reverse transcriptase PCR (RT-qPCR) assays for (i) the generic detection of the haemagglutinin (HA) gene of H7 viruses or the neuraminidase (NA) gene of N9 viruses, and (ii) the specific detection of HA and NA of the novel avian H7N9/2013 virus. The sensitivity of the newly developed assays was compared with previously published PCRs, and the specificity of all RT-qPCRs was examined using a panel of 42 different H7 and 16 different N9 isolates. Furthermore, we analysed the performance of the RT-qPCR assays with dilution series and diagnostic samples obtained from animal experiments. Our study provides a comprehensive set of RT-qPCR assays for the reliable detection of the novel avian H7N9 virus, with high sensitivity and improved and tailored specificity values compared with published assays. Finally, we also present data about the robustness of a duplex assay for the simultaneous detection of HA and NA of the avian influenza H7N9/2013 virus.

Towards a new, ecologically targeted approach to monitoring wild bird populations for avian influenza viruses.

Prevalence monitoring of avian influenza in wild bird populations is important to estimate risks for the occurrence of potentially zoonotic and economically disastrous outbreaks of highly pathogenic avian influenza virus (AIV) in poultry worldwide. A targeted, cost-effective monitoring method for AIV in wild birds was developed, which is based on monitoring results for AIV in Germany and information on the distribution and abundance of wild bird species in selected habitat types. Spatial data were combined with virological and outbreak data for the period of 1 January 2006 to 31 December 2010. Using Germany as an example, we identified 11 indicator species. By concentrating monitoring efforts on these species in spatially confined locations, we propose a targeted and more cost-effective risk-based AIV monitoring approach that can be adapted universally for the identification of wild bird indicator species worldwide with the perspective of reducing sample sizes (and costs) without impairing the validity of the results.

Consecutive natural influenza a virus infections in sentinel mallards in the evident absence of subtype-specific hemagglutination inhibiting antibodies.

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand-raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT-qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full-length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian-type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re-introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein-specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds.

Broad spectrum reactivity versus subtype specificity-trade-offs in serodiagnosis of influenza A virus infections by competitive ELISA.

Avian influenza viruses (AIVs) of the H5 and H7 subtypes can cause substantial economic losses in the poultry industry and are a potential threat to public health. Serosurveillance of poultry populations is an important monitoring tool and can also be used for control of vaccination campaigns. The purpose of this study was to develop broadly reactive, yet subtype-specific competitive ELISAs (cELISAs) for the specific detection of antibodies to the notifiable AIV subtypes H5 and H7 as an alternative to the gold standard haemagglutination inhibition assay (HI). Broadly reacting monoclonal competitor antibodies (mAbs) and genetically engineered subtype H5 or H7 haemagglutinin antigen, expressed and in vivo biotinylated in insect cells, were used to develop the cELISAs. Sera from galliform species and water fowl (n=793) were used to evaluate the performance characteristics of the cELISAs. For the H5 specific cELISA, 98.1% test sensitivity and 91.5% test specificity (97.7% and 90.2% for galliforms; 98.9% and 92.6% for waterfowl), and for the H7 cELISA 97.3% sensitivity and 91.8% specificity (95.3% and 98.9% for galliforms; 100% and 82.7% for waterfowl) were reached when compared to HI. The use of competitor mAbs with broad spectrum reactivity within an AIV haemagglutinin subtype allowed for homogenous detection with high sensitivity of subtype-specific antibodies induced by antigenically widely distinct isolates including antigenic drift variants. However, a trade-off regarding sensitivity versus nonspecific detection of interfering antibodies induced by phylo- and antigenically closely related subtypes, e.g., H5 versus H2 and H7 versus H15, must be considered. The observed intersubtype antibody cross-reactivity remains a disturbance variable in AIV subtype-specific serodiagnosis which negatively affects specificity.

Epidemiological and ornithological aspects of outbreaks of highly pathogenic avian influenza virus H5N1 of Asian lineage in wild birds in Germany, 2006 and 2007.

In Germany, two distinct episodes of outbreaks of highly pathogenic avian influenza virus of subtype H5N1 (HPAIV H5N1) in wild birds occurred at the beginning of 2006, and in summer 2007. High local densities of wild bird populations apparently sparked clinically detectable outbreaks. However, these remained restricted in (i) number of birds, (ii) species found to be affected, (iii) time, and (iv) location despite the presence of several hundred thousands of susceptible wild birds and further stressors (food shortage, harsh weather conditions and moulting). Northern and southern subpopulations of several migratory anseriform species can be distinguished with respect to their preference for wintering grounds in Germany. This corroborates viral genetic data by Starick et al. (2008) demonstrating the introduction of two geographically restricted virus subpopulations of Qinghai-like lineage (cluster 2.2.A and 2.2.B) into northern and southern Germany, respectively, in 2006. The incursion of virus emerging in 2007, found to be distinct from the clusters detected in 2006 (Starick et al., 2008), may have been associated with moulting movements. Intensive past-outbreak investigations with negative results of live and dead wild birds and of terrestrial scavengers excluded continued circulation of virus on a larger scale. However, persistence of virus in small pockets of local wild bird populations could not be ruled out resiliently. 1.5% of investigated sera originating from cats sampled at the epicentres of the Ruegen 2006-outbreak contained H5-antibodies. Passive monitoring was found to be highly superior to live bird surveillance when aiming at the detection of HPAIV H5N1 in wild birds (P < 0.0001).

Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses.

Accurate identification of hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A viruses is an integral part of monitoring programs targeting avian influenza viruses (AIV). Use of highly sensitive molecular screening methods such as pan influenza-specific real-time RT-PCR (rRT-PCR) yields an increasing number of samples which are positive for AIV RNA but negative by virus isolation and, therefore, require molecular, instead of serological, subtyping. We developed specific RT-PCR assays for all known nine AIV NA subtypes. Validation using 43 reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity. On basis of serial tenfold dilution series of reference isolates a benchmark value of C(t) 32 in an M gene-specific rRT-PCR became evident below which all nine NA subtypes were readily detectable by the subtype-specific RT-PCRs. For subtypes N1, N2, N4 and N6 detection was extended to dilutions with C(t) values of up to 35. Diagnostic applicability of the whole set of conventional NA-specific RT-PCRs was evaluated by analysis of 119 different diagnostic samples from wild birds which proved to be positive for AIV by M gene-specific rRT-PCR. Diagnostic sensitivity and specificity was confirmed by sequencing NA amplicons from 41 field isolates generated from this set and by NA inhibition assays. A universal molecular HA/NA subtyping algorithm for rRT-PCR positive avian influenza virus monitoring samples is proposed which may complement classical serological subtyping of influenza A virus isolates.

Phylogenetic analyses of highly pathogenic avian influenza virus isolates from Germany in 2006 and 2007 suggest at least three separate introductions of H5N1 virus.

In spring 2006, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was detected in Germany in 343 dead wild birds, as well as in a black swan (Cygnus atratus) kept in a zoo, three stray cats, one stone marten (Martes foina), and in a single turkey farm. In 2007 (June-July) the virus reoccurred in 96 wild birds at six geographically separate locations in the Southeast of Germany. In addition, a backyard mixed duck and goose holding was affected. Real-time RT-PCR [Hoffmann, B., Harder, T., Starick, E., Depner, K., Werner, O., Beer, M., 2007. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR. J. Clin. Microbiol. 45, 600-603] and nucleotide sequencing confirmed that these H5-viruses belonged to the Qinghai lineage of HPAIV H5N1 (clade 2.2). For a more detailed analysis, the hemagglutinin and neuraminidase genes of 27 selected German H5N1 viruses isolated 2006 or 2007 and originating from different regions and animal species were sequenced and analysed phylogenetically. As a result, three closely related but distinguishable H5N1 subclades could be defined: In 2006 a 'Northern type' (subclade 2.2.2), representing virus isolates from the German federal states Mecklenburg-Western Pomerania, Schleswig-Holstein, Brandenburg, and Lower Saxony, and a 'Southern type' (subclade 2.2.1) from Baden-Württemberg and Bavaria were detected. Interestingly, representatives of both types were present in Central Germany and caused the outbreak in turkeys (subclade 2.2.2) and in a case in a tufted duck (Aythya fuligula) (subclade 2.2.1) in Saxony. Furthermore, one isolate from the South of Germany was identified as 2.2.2 and vice versa a 2.2.1-like isolate was found in Northern Germany. H5N1 viruses isolated in 2007 belonged to a third type (subclade 2.2.3) which was not detected in 2006. Our data suggest the introduction of three distinct H5N1 variants into the wild bird population of Germany. The source of these viruses and the exact time of introduction remain obscure. Based on the identification of closely related H5N1 viruses from Southern and Central Russia, a recent introduction via wild birds on winter escape from these regions, early in 2006 constitutes the most likely scenario for the 2006 outbreaks. The viruses detected in 2007 most likely represent another new incursion from an as yet unknown source.

Isolation and characterization of a low-pathogenicity H7N7 influenza virus from a turkey in a small mixed free-range poultry flock in Germany.

A hemagglutinating virus was isolated from a dead turkey in a small mixed free-range flock in Southern Germany. It was identified as influenza virus type A of subtype H7N7. The pathogenicity was low. An intravenous pathogenicity index of 0.03 was recorded, and the nucleotide sequencing revealed the amino acid sequence NVPEIPKGR*GLFG at the cleavage site of the hemagglutinin. Antibodies as well as virus were detected in the affected flock. Further virus spreading to other flocks was prevented by stamping out policy. Serological monitoring of contact flocks revealed one small backyard flock of 18 hens, which was positive. This flock was also destroyed. The origin of the virus could not be identified.

Avian paramyxovirus serotype 1 isolates from the spinal cord of parrots display a very low virulence.

The spinal cord of 32 psittacines suffering from proventricular dilatation disease (PDD) was investigated. In six cases, a virus was isolated which upon electron microscopic examination revealed morphological details typical of members of the Paramyxoviridae. All isolates were subsequently characterized as avian paramyxovirus serotype 1 (APMV-1) by type-specific polyclonal antisera. According to their reactivity with APMV-1 specific monoclonal antibodies, the six isolates shared epitopes within the haemagglutinin-neuraminidase spike protein, distinct from pigeon-type paramyxoviruses and the LaSota vaccine strain. This grouping was further corroborated by properties of the haemagglutinin: all isolates showed a very thermosensitive haemagglutination activity and were rapid eluters. Virulence of the APMV-1 isolates in 1-day-old specific pathogen free (spf) chicken was very low, with intracerebral pathogenicity indices between 0 and 0.1. In embryonated spf chicken eggs, psittacine isolates replicated to high titres (10(8.6)-10(10.7) EID50/ml). However, they exhibited a reduced lethality over an observation time of 7 days (10(6.1)-10(8.3) ELD50/ml). In a haemagglutination inhibition test with parrot sera from birds with no history of APMV-1 vaccination, sera reacted preferentially with two isolates compared with APMV-1 vaccine strains LaSota and B1. The other four isolates exhibited a differentiated reaction pattern with the parrot sera, indicating an antigenic inhomogeneity. This is the first report of isolating very low virulent APMV-1 from neuronal tissue of parrots and implications for a possible role in slow progressing disease will be discussed.

Drebrin particles: components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia.

Drebrin, an actin-binding 70-kDa protein with an unusually slow SDS-PAGE mobility corresponding to approximately 120 kDa, containing a proline-rich, profilin-binding motif, had originally been reported from neuronal cells, but recently has also been found in diverse other kinds of tissues and cell lines. In biochemical analyses of various cells and tissues, employing gel filtration, sucrose gradient centrifugation, immunoprecipitation and -blotting, we have identified distinct states of soluble drebrin: a approximately 4S monomer, an 8S, ca. 217-kDa putative trimer, a 13S and a > 20S oligomer. In the 8S particles only [35S]methionine-labelled drebrin but no other actin-binding protein has been detected in stoichiometric amounts. By immunofluorescence and immunoelectron microscopy, drebrin-positive material often appeared as "granules" up to 400 nm in diameter, in some cell types clustered near the Golgi apparatus or in lamellipodia, particularly at leading edges, or in dense-packed submembranous masses at tips (acropodia) or ruffles of leading edges, in filopodia and at plaques of adhering junctions. We conclude that these drebrin complexes and drebrin-rich structures allow the build-up and maintenance of high local drebrin concentrations in strategic positions for the regulation of actin filament assembly, thereby contributing to cell motility and morphology, in particular local changes of plasticity and the formation of protrusions.

Formation of a normal epidermis supported by increased stability of keratins 5 and 14 in keratin 10 null mice.

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10(-/-) mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10(-/-) mice suggests that there is a considerable redundancy in the keratin gene family.

Supplementation of a mutant keratin by stable expression of desmin in cultured human EBS keratinocytes.

Mutations in keratin genes give rise to a number of inherited skin fragility disorders, demonstrating that the intermediate filament cytoskeleton has an essential function in maintaining the structural integrity of epidermis and its appendages. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder caused by mutations in keratins K5 or K14, which are expressed in the basal layer of stratified epithelia. Using a keratinocyte cell line established from an EBS patient, we investigated whether the muscle-specific intermediate filament protein desmin would be able to functionally complement a mutant keratin 14 in cultured keratinocytes. We show that in stably transfected EBS cells, desmin forms an extended keratin-independent cytoskeleton. Immunogold-EM analysis demonstrated that in the presence of numerous keratin filaments attached to desmosomes, desmin could nevertheless interact with desmosomes in the same cell, indicating the dynamic nature of the filament-desmosome association. When desmin-transfected cells were subjected to heat shock, the mutant keratin filaments showed a transient collapse while desmin filaments were maintained. Thus the defective keratin filaments and the wild-type desmin filaments appear to coexist in cells without interference. Expression of a type III intermediate filament protein like desmin may offer a strategy for the treatment of patients suffering from epidermal keratin mutations.

Newcastle disease and avian influenza A virus in wild waterfowl in South Africa.

In an intensive ostrich farming area in South Africa with a history of ostrich influenza outbreaks, we conducted a survey of avian influenza virus (AIV) and Newcastle disease virus (NDV) in wild aquatic birds. During late autumn and winter 1998, the time of year when outbreaks in ostriches typically start to occur, 262 aquatic birds comprising 14 species were sampled and tested for both virus infections. From eight samples, AIV, serotype H10N9, could be isolated. All isolates were apathogenic as determined by the intravenous pathogenicity index (0.00). Conversely, none of 33 sera of these wild birds showed antibodies against H10. However, one bird was found serologically positive for H6 AIV. This AIV serotype was later isolated from ostriches during an avian influenza outbreak in this area. No NDV was isolated although 34 of 46 serum samples contained NDV-specific antibodies. This is the first H10N9 isolate to be reported from Africa. In addition, our data support the notion that wild aquatic birds may function as a reservoir for AIV and NDV in South Africa.

H-1 parvovirus-associated replication bodies: a distinct virus-induced nuclear structure.

We have identified a nuclear structure that is induced after infection with the autonomous parvovirus H-1. Using fluorescence microscopy, we observed that the major nonstructural protein (NS1) of H-1 virus which is essential for viral DNA amplification colocalized with virus-specific DNA sequences and sites of ongoing viral DNA replication in distinct nuclear bodies which we designated H-1 parvovirus-associated replication bodies (H-1 PAR-bodies). In addition, two cellular proteins were shown to accumulate in H1 PAR-bodies: (i) the proliferating cell nuclear antigen (PCNA) which is essential for chromosomal and parvoviral replication and (ii) the NS1-interacting small glutamine-rich TPR-containing protein (SGT), suggesting a role for the latter in parvoviral replication and/or gene expression. Since many DNA viruses target preexisting nuclear structures, known as PML-bodies, for viral replication and gene expression, we have determined the localization of H-1 PAR- and PML-bodies by double-fluorescence labeling and confocal microscopy and found them to be spatially unrelated. Furthermore, H-1 PAR-bodies did not colocalize with other prominent nuclear structures such as nucleoli, coiled bodies, and speckled domains. Electron microscopy analysis revealed that NS1, as detected by indirect immunogold labeling, was localized in ring-shaped electron-dense nuclear structures corresponding in size and frequency to H-1 PAR-bodies. These structures were also clearly visible without immunogold labeling and could be detected only in infected cells. Our results suggest that H-1 virus does not target known nuclear bodies for DNA replication but rather induces the formation of a novel structure in the nucleus of infected cells.

Drebrin is a widespread actin-associating protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells.

Using immunoblotting, immunprecipitation with subsequent fragment mass spectrometry, and immunolocalization techniques, we have detected the actin-binding ca. 120-kDa protein drebrin, originally identified in - and thought to be specific for - neuronal cells, in diverse kinds of human and bovine non-neuronal cells. Drebrin has been found in numerous cell culture lines and in many tissues of epithelial, endothelial, smooth muscle and neural origin but not in, for example, cardiac, skeletal and certain types of smooth muscle cells, in hepatocytes and in the human epithelium-derived cell culture line A-431. By double-label fluorescence microscopy we have found drebrin enriched in actin microfilament bundles associated with plaques of cell-cell contact sites representing adhering junctions. These drebrin-positive, adhering junction-associated bundles, however, are not identical with the vinculin-containing, junction-attached bundles, and in the same cell both subtypes of microfilament-anchoring plaques are readily distinguished by immunolocalization comparing drebrin and vinculin. The intracellular distribution of the drebrin- and the vinculin-based microfilament systems has been studied in detail by confocal fluorescence laser scanning microscopy in monolayers of the polar epithelial cell lines, MCF-7 and PLC, and drebrin has been found to be totally and selectively absent in the notoriously vinculin-rich focal adhesions. The occurrence and the possible functions of drebrin in non-neuronal cells, notably epithelial cells, and the significance of the existence of two different actin-anchoring junctional plaques is discussed.

Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers.

A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus species and of the previously uncharacterized DNA polymerase genes of equine herpesvirus 3 (EHV-3) and equine herpesvirus 5 (EHV-5). The modified assay was then used for partial amplification of the polymerase of columbid herpesvirus 1 which is presently classified as a beta-herpesvirus based on biological criteria. Sequence analysis of amplicons obtained from four different viral strains revealed a close relationship of columbid herpesvirus 1 to members of the subfamily Alphaherpesvirinae, especially to Marek's disease herpesvirus. This was confirmed by characterization of additional 1.6kb of the columbid herpesvirus 1 polymerase. Consensus PCR analysis of blood samples from zebras, a wild ass and a tapir revealed amplicons showing high percentages ( > 50%) of sequence identity to DNA polymerases of gamma-herpesviruses. In particular, the zebra and the wild ass sequence were closely related to each other and to the polymerases of the equine gamma-herpesviruses EHV-2 and EHV-5 with sequence identities of > 80%. This is a first indication that novel gamma-herpesviruses are present in wild and zoo equids.