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.

Genetic characterisation of bovine herpesvirus 1 in New Zealand

AIM: To genotype bovine herpesvirus type 1 (BHV-1) isolates from cattle in New Zealand. METHODS: Twenty-eight BHV-1 isolates were collected from clinical samples from cattle over 28 years. They were characterised and compared using restriction endonuclease analysis (REA), and polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Twenty-four isolates were classified as bovine herpesvirus subtype 1.2b (BHV-1.2b) by REA. The remaining four isolates were distinct from the others in REA profiles of one of the major enzymes (HindIII) by which the classification was made. However, these four isolates were closely related to others when the REA profiles of other restriction enzymes were studied, and therefore were regarded as divergent strains of BHV-1.2b. All BHV-1 isolates were detectable by PCR, and sequence analysis of selected PCR products did not indicate any significant differences between isolates. CONCLUSION: BHV-1.2b appears to be the predominant strain of BHV-1 in cattle in New Zealand. There was no evidence that more virulent strains of BHV-1, e.g. subtype 1.1 and BHV type 5, are, or have been, present in New Zealand. Genetic variations exist among these BHV-1.2b isolates.

The essential and non-essential genes of Bovine herpesvirus 1

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Randominsertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.

Diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus 1 (infectious laryngotracheitis virus).

An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here.

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction.

A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.

The meleagrid herpesvirus 1 genome is partially resistant to transposition

The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed. © American Association of Avian Pathologists.

Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek’s disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek’s disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

The first genome sequence of a metatherian herpesvirus: Macropodid herpesvirus 1

While many placental herpesvirus genomes have been fully sequenced, the complete genome of a marsupial herpesvirus has not been described. Here we present the first genome sequence of a metatherian herpesvirus, Macropodid herpesvirus 1 (MaHV-1).

First detection of the equine herpesvirus 1 neuropathogenic variant in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

First defection of the equine herpesvirus 1 neuropathogenic variant in Brazil

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G 2254/D 752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A 2254/N 752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Comparative analysis of the replication of bovine herpesvirus 1 (BHV1) and BHV5 in bovine derived-neuron-like cells

Members of the subfamily Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as preferential sites for primary replication. However, bovine herpesvirus 5 (BoHV5) is neurotropic and neuroinvasive and responsible for meningoencephalitis in cattle and in animal models. A related virus, BoHV1 has also been occasionally implicated in natural cases of neurological infection and disease in cattle. The aim of the present study was to assess the in vitro effects of BoHV1 and BoHV5 replication in neuron-like cells. Overall, cytopathic effects, consisting of floating rounded cells, giant cells and monolayer lysis, induced by both viruses at 48 h postinfection (p.i.) resulted in a loss of cell viability and high virus titres (r = 0.978). The BoHV1 Cooper strain produced the lowest titres in neuron-like cells, although viral DNA was detected in infected cells during all experiments. Virus replication in infected cells was demonstrated by immunocytochemistry, flow cytometry and qPCR assays. BoHV antigens were better visualized at 48 h p.i. and flow cytometry analysis showed that SV56/90 and Los Angeles antigens were present at higher levels. In spite of the fact that BoHV titres dropped at 48 h p.i, viral DNA remained detectable until 120 h p.i. Sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and annexin V assays were used to identify apoptosis. BoHV5 induced death in approximately 50 % of cells within 24 h p.i., similar to what has been observed for BoHV1 Los Angeles. Infection with the BoHV1 Cooper strain resulted in 26.37 % of cells being in the early stages of apoptosis; 63.69 % of infected cells were considered viable. Modulation of mitochondrial function, as measured by mitochondrial membrane depolarization, was synchronous with the virus replication cycle, cell viability and virus titres at 48 h p.i. Our results indicate that apoptosis plays an important role in preventing neuronal death and provides a bovine-derived in vitro system to study herpesvirus-neuron interactions.