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Codon pair bias deoptimization (CPBD) has been successfully used to attenuate several RNA viruses. CPBD involves recoding a viral protein-coding sequence to maximize the number of codon pairs that are statistically underrepresented in the host, which presumably slows protein translation and, hence, causes virus attenuation. However, since recoding preserves the amino acid composition and codon bias, attenuated and parental viruses are antigenically identical. To determine if Marek's disease virus (MDV), a highly oncogenic herpesvirus of the chicken with a large double-stranded DNA genome, can be attenuated by CPBD of its major oncogene meq, we recoded the gene to minimize (meq-D), maximize (meq-O), or preserve (meq-R) the level of codon pairs that are overrepresented in the chicken protein-coding sequences. Unexpectedly, mutants carrying recoded genes produced comparable or increased levels of Meq in the context of viral infection in cultured cells. In addition, parental virus and mutant viruses carrying recoded meq genes replicated with comparable kinetics in vitro and in vivo, and were equally virulent in susceptible chickens. In summary, CPBD of meq failed to produce any quantifiable attenuation of MDV and confirms differences in the complexity of applying CPBD to large DNA viruses versus RNA viruses.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a high-priority pathogen in pandemic preparedness research. Reverse genetics systems are a valuable tool to study viral replication and pathogenesis, design attenuated vaccines and create defined viral assay systems for applications such as antiviral screening. Here we present a novel reverse genetics system for MERS-CoV that involves maintenance of the full-length viral genome as a cDNA copy inserted in a bacterial artificial chromosome amenable to manipulation by homologue recombination, based on the bacteriophage λ Red recombination system. Based on a full-length infectious MERS-CoV cDNA clone, optimal genomic insertion sites and expression strategies for GFP were identified and used to generate a reporter MERS-CoV expressing GFP in addition to the complete set of viral proteins. GFP was genetically fused to the N-terminal part of protein 4a, from which it is released during translation via porcine teschovirus 2A peptide activity. The resulting reporter virus achieved titres nearly identical to the wild-type virus 48 h after infection of Vero cells at m.o.i. 0.001 (1×10 5  p.f.u. ml -1 and 3×10 5  p.f.u. ml -1 , respectively), and allowed determination of the 50 % inhibitory concentration for the known MERS-CoV inhibitor cyclosporine A based on fluorescence readout. The resulting value was 2.41 µM, which corresponds to values based on wild-type virus. The reverse genetics system described herein can be efficiently mutated by Red-mediated recombination. The GFP-expressing reporter virus contains the full set of MERS-CoV proteins and achieves wild-type titres in cell culture.

Despite the importance of neurological disorders associated with herpesviruses, the mechanism by which these viruses influence the central nervous system (CNS) has not been definitively established. Owing to the limitations of studying neuropathogenicity of human herpesviruses in their natural host, many aspects of their pathogenicity and immune response are studied in animal models. Here, we present an important model system that enables studying neuropathogenicity of herpesviruses in the natural host. Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus that causes a devastating neurological disease (EHV-1 myeloencephalopathy; EHM) in horses. Like other alphaherpesviruses, our understanding of virus neuropathogenicity in the natural host beyond the essential role of viraemia is limited. In particular, information on the role of different viral proteins for virus transfer to the spinal cord endothelium in vivo is lacking. In this study, the contribution of two viral proteins, DNA polymerase (ORF30) and glycoprotein D (gD), to the pathogenicity of EHM was addressed. Furthermore, different cellular immune markers, including alpha-interferon (IFN-α), gamma-interferon (IFN-γ), interleukin-10 (IL-10) and interleukin-1 beta (IL-1β), were identified to play a role during the course of the disease.

Glycoprotein B (gB) of equine herpesvirus type 1 (EHV-1) is predicted to be cleaved by furin in a fashion similar to that of related herpesviruses. To investigate the contribution of furin-mediated gB cleavage to EHV-1 growth, canonical furin cleavage sites were mutated. Western blot analysis of mutated EHV-1 gB showed that it was cleaved at two positions, 518RRRR521 and 544RLHK547, and that the 28 aa between the two sites were removed after cleavage. Treating infected cells with either convertase or furin inhibitors reduced gB cleavage efficiency. Further, removal of the first furin recognition motif did not affect in vitro growth of EHV-1, while mutation of the second motif greatly affected virus growth. In addition, a second possible signal peptide cleavage site was identified for EHV-1 gB between residues 98 and 99, which was 13 aa downstream of that previously identified.

Equid herpesvirus type 1 (EHV-1) was shown to use an unusual receptor for cellular entry - MHC-I molecules. Here, we demonstrated that the closely related EHV, EHV-4, also uses this strategy for cellular invasion, both in equine cells in culture and in the heterologous, non-permissive murine mastocytoma cell line (P815) after stable transfection with horse MHC-I genes. Using a panel of P815 cell lines transfected with individual horse MHC-I genes, we provided support for the hypothesis that EHV-1 and EHV-4 target classical polymorphic MHC-I molecules as viral entry receptors. All known equine MHC-I molecules from the two principal classical polymorphic loci specify alanine at position 173 (A173), whilst other MHC-I loci encoded different amino acids at this position and did not permit viral entry. Site-directed mutagenesis of position 173 diminished or enhanced viral entry, depending upon the initial amino acid. However, there were other, as yet undefined, constraints to this process: MHC-I genes from two non-classical loci carried A173 but did not enable viral entry in P815 transfectants. Our study suggested that the capacity to bind MHC-I molecules arose in the common ancestor of EHV-1 and EHV-4. The widespread occurrence of A173 in classical polymorphic horse MHC-I molecules indicated that horses of most MHC haplotypes should be susceptible to infection via this entry portal., (© 2014 The Authors.)

Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle that is maintained in the population by persistently infected animals. Virus infection may result in reproductive failure, respiratory disease and diarrhoea in naïve, susceptible bovines. Here, the construction and characterization of a novel vectored vaccine, which is based on the incorporation of genes encoding BVDV structural proteins (C, Erns, E1, E2) into a bacterial artificial chromosome of the equine herpesvirus type 1 (EHV-1) vaccine strain RacH, are reported. The reconstituted vectored virus, rH_BVDV, expressed BVDV structural proteins efficiently and was indistinguishable from parental vector virus with respect to growth properties in cultured cells. Intramuscular immunization of seronegative cattle with rH_BVDV resulted in induction of BVDV-specific serum neutralizing and ELISA antibodies. Upon experimental challenge infection of immunized calves with the heterologous BVDV strain Ib SE5508, a strong anamnestic boost of the neutralizing-antibody response was observed in all vaccinated animals. Immunized animals presented with reduced viraemia levels and decreased nasal virus shedding, and maintained higher leukocyte counts than mock-vaccinated controls.

The genome of bovine herpesvirus type 1 Schönböken was cloned as a bacterial artificial chromosome (BAC) by inserting mini F plasmid sequences into the glycoprotein (g) E gene. The resulting BAC clone, pBHV-1DeltagE, was transfected into bovine kidney cells and viable gE-negative BHV-1 (BHV-1DeltagE) was recovered. By RecE/T mutagenesis in Escherichia coli, the gG open reading frame was deleted from pBHV-1DeltagE. From the mutated BAC, double negative BHV-1DeltagE-gG was reconstituted and its growth properties were compared to those of rescuant viruses in which the gE gene was restored (BHV-1rev, BHV-1DeltagG). The mutant viruses did not exhibit markedly lowered virus titres. Plaque sizes of BHV-1DeltagE, BHV-1DeltagE-gG and BHV-1DeltagG, however, were reduced by 19 to 55 % compared to parental strain Schönböken or BHV-1rev. Our results suggested that gE and gG function independently from each other in cell-to-cell spread, because an additive effect on plaque formation was observed in the gE/gG double deletion mutant.

A DNA vaccine containing the infectious BAC20 clone of serotype 1 Marek's disease virus (MDV) was tested for its potential to protect against Marek's disease (MD). Chickens were immunized at 1 day old with BAC20 DNA suspended either in PBS, as calcium phosphate precipitates, incorporated into chitosan nanoparticles, in Escherichia coli DH10B cells, or bound to gold particles for gene-gun delivery. Challenge infection with MDV strain EU1 was performed at 12 days old, and four out of seven birds immunized with BAC20 DNA in saline by the intramuscular route remained free of MD until day 77 after challenge infection. A delay in the development of the disease could be observed in some animals vaccinated with other BAC20 DNA formulations, but clinical MD and tumour formation were evident in all but one bird. Five out of seven animals immunized with the vaccine virus CVI988 were protected against MD, but none out of seven birds survived EU1 challenge infection after injection of negative-control plasmid DNA. In a second animal experiment, five out of 12 chickens immunized with BAC20 DNA and six out of eight birds immunized with virus reconstituted from BAC20 DNA remained free of MD after challenge infection. In contrast, none out of 12 chickens survived challenge infection after immunization with BAC20 DNA lacking the essential gE gene or with gE-negative BAC20 virus. The results suggested that an MDV BAC DNA vaccine has potential to protect chickens against MD, but that in vivo reconstitution of vaccine virus is a prerequisite for protection.

A recombinant cell line (SOgE) was established, which was derived from the permanent quail muscle cell line QM7 and constitutively expressed the glycoprotein E (gE) gene of Marek's disease virus serotype 1 (MDV-1). The SOgE cell line supported growth of virulent (RB-1B) and vaccine (CVI988, 584Ap80C) MDV-1 strains at a level comparable with that of primary chicken embryo cells (CEC). The SOgE cell line was used to produce a vaccine against Marek's disease. Chickens were immunized at 1 day old with 10(3) p.f.u. CVI988 produced on either CEC or SOgE cells. Challenge infection was performed at day 12 with hypervirulent Italian MDV-1 strain EU1. Whereas 7/7 or 6/6 animals, respectively, immunized with SOgE or QM7 cells alone developed Marek's disease, only 1/8 animals from both CVI988-immunized groups exhibited signs of disease, suggesting that SOgE cells are a valuable permanent cell culture system for MDV-1 vaccine production.

The role of the products of the UL10 and the UL49.5 homologous genes of Marek's disease virus serotype 1 (MDV-1) in virus replication was investigated. Deletion of either open reading frame in an infectious bacterial artificial chromosome clone (BAC20) of MDV-1 resulted in progeny viruses that were unable to spread from cell to cell. After transfection of UL10- or UL49.5-negative BAC20 DNA into chicken or quail cells, only single infected cells were observed by indirect immunofluorescence analysis. In contrast, plaque formation was restored when mutant BAC DNAs were co-transfected with the corresponding expression plasmid encoding either the UL10-encoded gM or the UL49.5 gene product. These data demonstrate that gM and its putative complex partner, the UL49.5 homologous protein, are essential for MDV-1 growth in cultured cells. Thus, MDV-1 represents the first example of a member of the family Herpesviridae for which the highly conserved membrane proteins are indispensable for cell-to-cell spread.