Background
Meleagrid herpesvirus 1 (MeHV-1), commonly known as turkey herpesvirus (HVT), is a non-pathogenic avian herpesvirus originally isolated from turkeys in 1969 [
1,
2]. The virus is assigned to the genus
Mardivirus, which also includes oncogenic
Gallid herpesvirus 2 (GaHV-2), the causative agent of Marek’s disease (MD), and the non-oncogenic
Gallid herpesvirus 3 (GaHV-3). Marek’s disease is a highly contagious neoplastic poultry disease of major economic significance worldwide. The close antigenic relationship amongst the mardiviruses has been exploited since the 1970s through the use of MeHV-1 as a live vaccine to reduce production losses resulting from MD [
3]. However, despite widespread vaccination with either MeHV-1, bivalent vaccines containing MeHV-1/GaHV-3 or attenuated GaHV-2 strains, MD outbreaks continue to occur. The isolation of GaHV-2 field strains of increased virulence has been correlated with the loss of protective capacity of these vaccines, which reinforces the need for development of improved MD vaccines [
4,
5]. It is likely that novel vaccines targeting GaHV-2 will be constructed using recombinant DNA technologies, for which MeHV-1 is ideally suited as a vector candidate.
In addition to its use as a vaccine against MD, MeHV-1 is also widely utilised as a recombinant vaccine vector for poultry diseases such as infectious laryngotracheitis, Newcastle disease, infectious bursal disease and highly pathogenic avian influenza [
6‐
9]. Currently, only a limited number of transgene insertion sites are used in the development of recombinant MeHV-1 based vaccines. Use of a suboptimal insertion site can have a pronounced effect on vaccine efficacy. For example, Gao
et al. [
10] reported a reduced post-challenge mortality with highly pathogenic avian influenza virus when the haemagglutinin gene was expressed from the MeHV-1
US2 locus compared to the
US10 locus, likely because
in vivo virus replication was more affected with the disruption of
US10 compared to
US2. Thus the identification of alternative transgene insertion sites will be useful for the optimisation of MeHV-1 as a vaccine vector.
The MeHV-1 genome is 159,160 bp in length and has a type 4 herpesvirus genomic structure [
11,
12]. It comprises a unique long (U
L) and a unique short (U
S) region, flanked by terminal/internal repeat long (TR
L/IR
L) and short segments (TR
S/IR
S), respectively [
11,
12]. In addition, the genomic termini comprise telomeric repeats, or a-like sequences, of variable length, which are cis-acting elements involved in genome packaging [
13]. These a-like sequences are also present at the IR
L/IR
S junction [
11]. For comparative purposes, this article refers to individual genes by their putative human herpesvirus 1 (HHV-1) homologue, using U
L and U
S nomenclature [
14]. Genes unique to mardiviruses are identified by their protein designation, as described in the MeHV-1 reference sequence [Genbank: NC_002641.1] [
11]. The complete MeHV-1 genome encodes 79 putative genes [
12]. Of these, 73 are single copy; 66 within the U
L region and seven in the U
S region. The genes
vNR13 and
icp4 are duplicated, with one copy of each located in the IR
S and TR
S elements. The
US8 gene, encoding the envelope glycoprotein E (gE), spans the TR
S-U
S boundary region; consequently, the gene
US8*, located within the IR
S, is a truncated duplication of
US8. Current knowledge of MeHV-1 gene function has largely been extrapolated from studies on GaHV-2, and more broadly from genetic studies of HHV-1 and other herpesviruses. While MeHV-1 is currently utilised as a vaccine vector, a more detailed understanding of the genetic background of this virus is required to facilitate its further development as a vector.
The establishment of infectious bacterial artificial chromosome (iBAC) technologies for herpesvirus mutagenesis has simplified the process of generating modified viruses for functional studies and recombinant vaccine construction. For global genome mutagenesis studies, transposition has previously been proven to be a valuable tool, since the random insertion of transposon sequences allows for the efficient generation of a library of unique insertional mutants. These mutants can then be screened to determine if the transposon insertion affects the replication capacity of the virus in cell culture [
15‐
19]. In this way, non-essential genetic loci can be readily identified, and concurrently tested for their potential to carry transgenes for the subsequent generation of recombinant vaccines.
The aim of this study was to characterise a MeHV-1 iBAC transposition library by determining the site of transposon insertion and the impact on viral replication in cell culture. Overall, twenty non-essential loci were identified within the MeHV-1 genome. Additionally, the requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time.
Discussion
The requirements for 28 coding and six non-coding regions within the MeHV-1 genome have been determined in this study, using a library of 76 transposition mutants. These included 11 genes for which the
in vitro requirements for replication have not previously been reported in any of the mardiviruses (Table
1). Clones with unique phenotypes and gene designations that contrast with the reported literature are discussed in detail below. Of particular interest in this study were those insertion sites that were non-essential for replication, as these represent potential transgene insertion sites for MeHV-1-based vectors. These sites included 14 intragenic and six intergenic sites.
Homologues of the
LORF2 gene are restricted to the
Mardivirus genus. It has been suggested that this gene may have a role in mRNA transcription or processing but this is yet to be experimentally confirmed [
12]. Recently, the GaHV-2
LORF2 homologue was reported to have immunoevasion functions via the down-regulation of MHC class I in infected cells [
23].
LORF2 has previously been reported as essential for GaHV-2 replication in cell culture [
18]. In contrast, another study reported retroviral insertions within the ORF as having no effect on GaHV-2 replication [
23,
24]. In the current study, transposition clone MuAΔ30 contained an insertion within the first exon of MeHV-1
LORF2, disrupting 99.5 % of the predicted polypeptide. Viral recovery experiments clearly demonstrated that this location was non-essential for replication of MeHV-1 in cell culture, with CPE developing within five days of transfection. The differing requirement of this gene in MeHV-1 and GaHV-2 is of interest, and further investigations into LORF2 function in MeHV-1 are warranted. Furthermore, the use of
LORF2 as a transgene insertion site for vaccine development may increase vaccine efficacy through impairment of the proposed LORF2 immunoevasion functions.
The non-essential classification of MeHV-1
UL10 in this study contrasts with the essential assignment of the GaHV-2
UL10 homologue (Table
1) [
25]. The
UL10 gene encodes a homologue of glycoprotein M (gM), a core herpesvirus gene [
11,
12]. The
UL10 gene is essential in the strictly cell-associated viruses GaHV-2 and
Human herpesvirus 3 (HHV-3) [
25,
26]. In contrast,
UL10 has consistently been reported as non-essential for viral replication in cell culture for cell-free herpesviruses such as HHV-1,
Suid herpesvirus 1 (SuHV-1),
Bovine herpesvirus 1 (BoHV-1),
Equine herpesvirus 1 (EHV-1) and
Gallid herpesvirus 1 [
19,
27‐
30]. Although the parental MeHV-1 strain FC126 used in this study was cell-associated, cell-free virus is produced to a limited extent and this strain can be adapted to produce high titres of cell-free virus [
31]. It has also been suggested that expression of glycoprotein D (gD) may compensate for loss of gM function, since both HHV-3 and GaHV-2 do not express gD in cell culture, and this may explain the essential designation of gM in these viruses [
25]. While the expression of MeHV-1 gD during infection in cell culture has not been reported to date, the capacity of the virus to adapt to cell-free growth suggests it is.
The
UL21 gene encodes a poorly characterised tegument protein that is capsid-associated and may have roles in intracellular transport and in nuclear egress [
32,
33]. The recovery of infectious MeHV-1 from three
UL21 transposed clones, Tn5Δ14, MuAΔ37 and MuAΔ41, in combination with the presence of viral DNA after sequential passages, confirms that MeHV-1
UL21 is non-essential for replication in cell culture. However, replication was severely attenuated compared to the parent virus. Disruption studies in other alphaherpesviruses have shown
UL21 to be non-essential, although a range of deleterious effects have been noted on virus replication (Table
1). A
UL21 mutant of SuHV-1 showed impaired replication in cell culture and reduced virulence
in vivo [
17,
34]. For HHV-1 and BoHV-1,
UL21 has been shown to be non-essential, but deletion reduced the
in vivo replication capacity of HHV-1 [
19,
35,
36]. In contrast,
UL21 has been reported to be essential for
Human herpesvirus 2, HHV-3 and EHV-1 [
26,
32,
37]. The severe attenuation observed for MeHV-1 in this study suggests the
UL21 gene is unsuitable for use in recombinant vaccine applications, however it may be of use for generating replication-limited gene delivery constructs for poultry research applications.
The non-essential phenotype of MeHV-1
UL48 disruption mutants characterised in this study conflicts with the essential requirement of this gene for HHV-1 replication (Fig.
1c and
1d). In HHV-1,
UL48 encodes the VP16 α-trans inducing factor, a tegument protein that induces immediate-early gene transcription and is also required for virion assembly [
38]. The
UL48 homologues of many alphaherpesviruses, including mardiviruses, lack the acidic carboxyl terminus transactivating domain present in the HHV-1
UL48 protein, however transactivating functions may be retained via other transactivation sites within
UL48 [
11,
12,
39,
40]. This gene is essential for the replication of HHV-1 and EHV-1 in cell culture, but is non-essential in other alphaherpesviruses investigated to date, including HHV-3, SuHV-1, BoHV-1 and GaHV-2 (Table
1) [
19,
26,
38,
41‐
45].
The MeHV-1
UL53 gene is a homologue of the HHV-1 gene encoding glycoprotein K (gK) [
11,
12]. Similar to other viral glycoproteins, gK has roles in cell-to-cell fusion and in viral egress from infected cells [
46,
47]. It has been reported to be essential for replication of many alphaherpesviruses, including GaHV-2, HHV-3, SuHV-1 and BoHV-1 (Table
1) [
19,
26,
48,
49], while it is non-essential for HHV-1 and EHV-1 growth
in vivo [
50,
51]. Interestingly, deletion of
UL53 from both the HHV-1 and EHV-1 genomes resulted in severely attenuated viruses with greatly reduced plaque sizes and impaired virion penetration in cell culture [
37,
51,
52]. Marked attenuation was also observed for the MeHV-1
UL53 disruption mutant, MuAΔ68, in this study (Fig.
1e and
1f).
The MeHV-1
LORF4A gene is a homologue of
LORF4 genes of GaHV-2 and GaHV-3 and
LORF9 of
Anatid herpesvirus 1, and the encoded polypeptide shares 47 % amino acid identity to the proposed paralogue, MeHV-1 LORF4 [
11,
12]. The
LORF4 homologues have been postulated to have roles as avian host range determinants, since the occurrence of this gene is restricted to mardiviruses [
11,
12]. Transposon insertion into
LORF4A in the pMeHV1-C18 transposon clone Tn5Δ10 disrupted 82 % of the gene, and the insertion location was designated as non-essential for virus growth in cell culture. This is the first report of a disruption mutant of
LORF4.
Overall fourteen genetic locations were identified as essential for MeHV-1 replication in cell culture (Table
1)
. The classification of these loci provides additional foundational information concerning MeHV-1 replication, as the requirements of 13 of these genes have not previously been reported for MeHV-1. Although it would have been interesting to determine the effects of insertions on global viral gene expression and protein production, this was beyond the scope of the current study. Similarly, revertant constructs were not generated for replication-defective mutants since putatively essential genes are not of further interest for vaccine development.
It is noteworthy that the transposition mutants reported here are cumulative gene deletion mutants of MeHV-1, as pMeHV1-C18 lacks seven coding regions compared to the parental virus [
20]. This genetic background may have contributed to the observed attenuation of some clones compared to the wild-type MeHV-1. It is considered unlikely that the requirement of the non-essential loci identified in this study would be essential in the full-length virus, as it is reasonable to conclude that effects on viral replication are likely to be more severe with cumulative gene deletions compared to the disruption of a single gene. However, it is possible the locations designated as essential in this study may be non-essential in the parental virus. Nonetheless, this is also considered unlikely as the MeHV-1 genes designated as essential in this study conform with the reported requirements for the respective homologues of other alphaherpesviruses, with the exception of
UL19 which is reported as non-essential in SuHV-1 (Table
1) [
17]. However, it must be noted that in that study, the transposon insertion event mapped 2 bp downstream of the SuHV-1
UL19 ORF, therefore it could be argued that this was not a true report of the UL19 requirement in this virus as complete translation of the encoded polypeptide would have been possible.
Given the instability observed in the LORF5 insertion mutants, it is possible that this is an essential gene and it may have been misclassified as non-essential in the current study. This is considered unlikely, since in the case of the LORF5 mutants, the transgene was gradually lost during serial passage of recovered virus. In the case of an insertion into an essential gene, the insertion mutant would not undergo sufficient replication capacity to facilitate loss of the transgene and subsequent recovery of virus. Regardless of whether the LORF5 gene is essential or non-essential for replication, the observed instability of the transposon insertions in two independent LORF5 transposon insertion mutants suggests this region of the genome would be unsuitable for recombinant vector applications.
Despite the potential limitations of the cumulative gene deletion genotype of the iBAC used in this study, it has enabled the identification of viruses with novel phenotypes, for example the MuAΔ68 virus with an insertion into
UL53. While CPE was observed, it was subtle compared to the parent virus and may have been missed completely in the absence of reporter gene expression (Fig.
1e and
1f). It is considered highly unlikely that a virus with this phenotype could be constructed using rational gene-targeting strategies.
Importantly, potential insertion sites for vector development must also be verified
in vivo, since it is generally accepted that non-essential genes in cell culture may have major roles
in vivo, for example in immunoevasion or other virus-host interactions [
14]. An example of this are glycoprotein C (gC)-null mutants of GaHV-2, which show increased viral replication in cultured cells
, however
in vivo infection required a longer incubation period to establish infection, viraemia and induction of seroconversion compared to gC-positive virus, and gC-null viruses were not transmitted horizontally [
53,
54]. The
in vivo replication capacity of virus recovered from the parental iBAC used in this study is reduced compared to wild-type MeHV-1 [
20]. As a result it might be expected that any constructs derived from this parent clone would be further attenuated
in vivo. Extrapolating from the GaHV-2 studies discussed above, a deletion identified in the
UL44 region of pMeHV1-C18 likely contributes to the
in vivo attenuation observed with this construct. Therefore consideration should be given to the restoration of this deletion prior to
in vivo assessment of the non-essential gene mutants constructed in the current study.
The strategy used to determine the replication requirement for icp4 highlights the power of iBAC technologies, for example to generate a dual-disruption mutant with two mutagenised copies of a repeat element. This strategy was developed after the generation of the transposition libraries, thus the presence of suitable RE sites in the Tn5 transposon and the virus was serendipitous. Future studies investigating genetic elements located in the repeat sequences of herpesvirus iBACs should consider the identification of suitable RE sites within the targeted viral genome to enable the identification of modified specific repeat sequences. If appropriate sites are identified in the virus, complementary sites could be readily incorporated into the proposed transgene molecule to facilitate the isolation of double-deletion/disruption mutants.
Authors’ contributions
RNH participated in design of the study, carried out the molecular studies and drafted the manuscript. JM and EVF participated in design of the study and critical revision of the manuscript. TJM conceived the study, and participated in its design and coordination and assisted with drafting the manuscript. All authors read and approved the final manuscript.