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Abstract Cloning of full length genomes of herpesviruses as bacterial artificial chromosomes (BAC) has greatly facilitated the manipulation of the genomes of several herpesviruses to identify the pathogenic determinants. We have previously reported the construction of the BAC clone (pRB-1B5) of the highly oncogenic Marek's disease virus (MDV) strain RB-1B, which has proven to be a valuable resource for elucidating several oncogenic determinants. Despite the retention of the BAC replicon within the genome, the reconstituted virus was able to induce tumours in susceptible chickens. Nevertheless, it was unclear whether the presence of the BAC influenced the full oncogenic potential of the reconstituted virus. To maximize the closeness of BAC-derived virus to the parental RB-1B strain, we modified the existing pRB-1B5 clone by restoring the Us2 and by introducing SV40-cre cassette within the loxP sites of the mini-F plasmid, to allow self-excision of the plasmid sequences in chicken cells. The reconstituted virus from the modified clone showed significant improvement in replication in vitro and in vivo. Excision of the BAC sequences also enhanced the pathogenicity to levels similar to that of the parental virus, as the cumulative incidence of Marek's disease in groups infected with the recombinant and the parental viruses showed no significant differences. Thus, we have been able to make significant improvements to the existing BAC clone of this highly oncogenic virus which would certainly increase its usefulness as a valuable tool for studies on identifying the oncogenic determinants of this major avian pathogen.

Respiratory syncytial virus (RSV) infection and shingles are two viral diseases that affect older adults, and a combined vaccine to protect against both could be beneficial. RSV infection causes hospitalisations and significant morbidity in both children and adults and can be fatal in the elderly. The RSV fusion (F) envelope glycoprotein induces a strong RSV-neutralising antibody response and is the target of protective immunity in the first RSV vaccine for older adults, recently approved by the FDA. An initial childhood infection with the varicella zoster virus (VZV) results in chickenpox disease, but reactivation in older adults can cause shingles. This reactivation in sensory and autonomic neurons is characterized by a skin-blistering rash that can be accompanied by prolonged pain. The approved protein-in-adjuvant shingles vaccine induces VZV glycoprotein E (gE)-fspecific antibody and CD4 + T cell responses and is highly effective. Here we report the evaluation of RSV/shingles combination vaccine candidates based on non-replicating chimpanzee adenovirus (ChAd) vectors. We confirmed the cellular and humoral immunogenicity of the vaccine vectors in mice using T cell and antibody assays. We also carried out an RSV challenge study in cotton rats which demonstrated protective efficacy following a homologous prime-boost regimen with our preferred vaccine candidate.

A response to Pervasive sequence patents cover the entire human genome by J Rosenfeld and C Mason. Genome Med 2013, 5:27. See related Correspondence by Rosenfeld and Mason, http://genomemedicine.com/content/5/3/27 and related letter by Rosenfeld and Mason, http://genomemedicine.com/content/6/2/15.

Duck enteritis virus (DEV) is an important herpesvirus pathogen associated with acute, highly contagious lethal disease in waterfowls. Using a deep sequencing approach on RNA from infected chicken embryo fibroblast cultures, we identified several novel DEV-encoded micro (mi)RNAs. Unlike most mardivirus-encoded miRNAs, DEV-encoded miRNAs mapped mostly to the unique long region of the genome. The precursors of DEV miR-D18 and miR-D19 overlapped with each other, suggesting similarities to miRNA-offset RNAs, although only the DEV-miR-D18-3p was functional in reporter assays. Identification of these novel miRNAs will add to the growing list of virus-encoded miRNAs enabling the exploration of their roles in pathogenesis.

The identification of specific genetic changes associated with differences in the pathogenicity of Marek's disease virus strains (GaHV-2) has been a formidable task due to the large number of mutations in mixed-genotype populations within DNA preparations. Very virulent UK isolate C12/130 induces extensive lymphoid atrophy, neurological manifestations and early mortality in young birds. We have recently reported the construction of several independent full-length bacterial artificial chromosome (BAC) clones of C12/130 capable of generating fully infectious viruses with significant differences in their pathogenicity profiles. Two of these clones (vC12/130-10 and vC12/130-15), which showed differences in virulence relative to each other and to the parental strain, had similar replication kinetics both in vitro and in vivo in spite of the fact that vC12/130-15 was attenuated. To investigate the possible reasons for this, the nucleotide sequences of both clones were determined. Sequence analysis of the two genomes identified mutations within eight genes. A single 494 bp insertion was identified within the genome of the virulent vC12/130-10 clone. Seven non-synonymous substitutions distinguished virulent vC12/130-10 from that of attenuated vC12/130-15. By sequencing regions of parental DNA that differed between the two BAC clones, we confirmed that C12/130 does contain these mutations in varying proportions. Since the individual reconstituted BAC clones were functionally attenuated in vivo and derived from a single DNA source of phenotypically very virulent C12/130, this suggests that the C12/130 virus population exists as a collection of mixed genotypes.

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

MDV-GX0101 is a field strain of Marek's disease virus with a naturally occurring insertion of the reticuloendotheliosis virus (REV) LTR fragment. In order to study the biological properties of REV-LTR insertion in the MDV genome, we constructed a full-length infectious BAC clone of MDV-GX0101 strain and deleted the LTR sequences by BAC mutagenesis. The pathogenic properties of the LTR-deleted virus were evaluated in infected SPF birds. The study demonstrated that the LTR-deleted virus had a stronger inhibitory effect on the growth rates of the infected birds and induced stronger immunosuppressive effects. Surprisingly, however, the ability for horizontal transmission of the LTR-deleted virus appeared to be significantly weaker than its parental LTR-intact virus. Even though the precise molecular mechanisms are still not clear, the results of our studies demonstrate that the retention of the REV-LTR in the MDV genome decreases its pathogenic effects but increases its potential for horizontal transmission., (Copyright 2009 Elsevier Inc. All rights reserved.)

We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5' leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.

Cloning of full length genomes of herpesviruses as bacterial artificial chromosomes (BAC) has greatly facilitated the manipulation of the genomes of several herpesviruses to identify the pathogenic determinants. We have previously reported the construction of the BAC clone (pRB-1B5) of the highly oncogenic Marek's disease virus (MDV) strain RB-1B, which has proven to be a valuable resource for elucidating several oncogenic determinants. Despite the retention of the BAC replicon within the genome, the reconstituted virus was able to induce tumours in susceptible chickens. Nevertheless, it was unclear whether the presence of the BAC influenced the full oncogenic potential of the reconstituted virus. To maximize the closeness of BAC-derived virus to the parental RB-1B strain, we modified the existing pRB-1B5 clone by restoring the Us2 and by introducing SV40-cre cassette within the loxP sites of the mini-F plasmid, to allow self-excision of the plasmid sequences in chicken cells. The reconstituted virus from the modified clone showed significant improvement in replication in vitro and in vivo. Excision of the BAC sequences also enhanced the pathogenicity to levels similar to that of the parental virus, as the cumulative incidence of Marek's disease in groups infected with the recombinant and the parental viruses showed no significant differences. Thus, we have been able to make significant improvements to the existing BAC clone of this highly oncogenic virus which would certainly increase its usefulness as a valuable tool for studies on identifying the oncogenic determinants of this major avian pathogen.

The complete DNA sequence of the Marek's disease virus serotype 1 vaccine strain CVI988 was determined and consists of 178 311 bp with an overall gene organization identical to that of the oncogenic strains. In examining open reading frames (ORFs), nine differ between vaccine and oncogenic strains. A 177 bp insertion was identified in the overlapping genes encoding the Meq, RLORF6 and 23 kDa proteins of CVI988. Three ORFs are predicted to encode truncated proteins. One, designated 49.1, overlaps the gene encoding the large tegument protein UL36 and encodes a severely truncated protein of 34 aa. The others, ORF5.5/ORF75.91 and ORF3.0/78.0, located in the repeat regions (diploid), encode a previously unidentified ORF of 52 aa and a truncated version of the virus-encoded chemokine (vIL-8), respectively. Subtle genetic changes were identified in the two ORFs encoding tegument proteins UL36 and UL49. Only one diploid ORF (ORF6.2/ORF75.6) present in the genomes of the three virulent strains is absent in the CVI988-BAC genome. Seventy non-synonymous amino acid substitutions were identified that could differentiate CVI988-BAC from all three oncogenic strains collectively. Estimates of the non-synonymous to synonymous substitution ratio (omega) indicate that CVI988 ORFs are generally under purifying selection (omega<1), whereas UL39, UL49, UL50, RLORF6 and RLORF7 (Meq) appear to evolve under relaxed selective constraints. No CVI988 ORF was found to be under positive evolutionary selection (omega>>1).

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the U(S)2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.

Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.