Background
Marek’s disease virus (MDV; family, Herpesviridae; subfamily, α-Herpesvirinae; genus,
Mardivirus; species,
Gallid alphaherpesvirus 2) is the causative agent of Marek’s disease (MD), which manifests as malignant lymphomas in infected chickens [
1]. MD previously caused serious economic losses to the poultry industry, but the introduction of vaccines has led to its successful control [
2]. Attenuated strains of MDV and the naturally non-oncogenic
Gallid alphaherpesvirus 3 and
Meleagrid alphaherpesvirus 1 (turkey herpesvirus, HVT) have been used as monovalent or multivalent vaccines. An attenuated MDV strain, CVI988, is considered the most protective vaccine currently available and has been introduced in many countries [
3]. However, the virulence of MDV field strains has a tendency to increase, and pathogenic MDV strains are generally classified as mild, virulent, very virulent, and very virulent+ [
4]. Currently, sporadic occurrences of MD are still reported in some countries [
4‐
11], and highly virulent MDV strains could potentially cause future outbreaks despite vaccination [
4].
The Meq protein, which is highly expressed in MDV-transformed cell lines and tumor samples [
12‐
15], is an oncoprotein of MDV. Its structure consists of an N-terminal basic region leucine zipper (bZIP) domain and a C-terminal transactivation domain. The bZIP domain, similar to that of the Jun/Fos family of oncoproteins, consists of two stretches of basic residues (basic regions 1 and 2) and a leucine zipper, whereas the transactivation domain is characterized by proline-rich repeats that contain several SH3-binding motifs [
16]. Meq can form dimers via the leucine zipper and interact with the promoter region of target genes through the basic region, thereby regulating gene expression in host cells and MDV [
16]. In addition, Meq can interact with p53 [
17] and the C-terminal binding protein [
18], ultimately affecting characteristics of transformation such as anti-apoptotic effects and regulation of gene expression. Thus, Meq plays important roles in transformation induced by MDV and is essential for viral pathogenesis.
Genetic approaches have revealed polymorphisms in Meq proteins among MDV strains isolated in USA and that they are associated with MDV virulence [
19]. Amino acid substitutions can affect Meq protein functions, transactivation activities, and transformation [
20,
21]. The evolution of
meq genes is comparable with the evolutionary rate of RNA viruses, and the time of
meq gene divergence is related to the transitions in management practices in the poultry industry, including the introduction of vaccines [
23]. Thus, the positive selection induced by vaccination seems to be a reason for the emergence of genetic diversity in the
meq gene [
23]. To date,
meq gene sequences in several regions other than the USA, including China, Europe and Australia, have been investigated [
5,
24‐
31]. The
meq genes in MDV strains isolated in each region were found to exhibit different genetic characteristics [
23]. Thus,
meq gene polymorphisms or evolution seem to reflect the geographical characteristics and the history of vaccine use in each region.
Whole-genome sequences of MDV strains isolated in some regions have been reported from the 2000s. The attenuated MDV vaccine strain, CVI988 [
32], MDV strains with different virulence in the USA [
33‐
38], pathogenic and vaccine strains in China [
39‐
44], and a pathogenic strain in Europe [
45,
46] were sequenced. Phylogenetic analysis indicated that most MDV strains can be classified into two clusters, namely, the North American or Eurasian clusters, and the estimated time-scaled phylogeny suggested that MDV virulence evolved independently in Eurasia and North America [
46]. CVI988 was initially isolated and developed in Europe as a vaccine and is used worldwide. However, in the USA, HVT was initially used for the prevention of MD, and a bivalent vaccine comprising HVT and SB-1 (naturally non-oncogenic
Gallid alphaherpesvirus 3) has been adopted after outbreaks in HVT-vaccinated chickens. Therefore, the history of vaccine control seems to affect the differential evolution of MDV strains in each continent [
46].
In Japan, MD occurrences are sporadic. We previously reported the sequences of
meq genes in MDV isolates in Japan and that the amino acid sequence at position 176 is serine or threonine in the most recent field isolates [
22]. This amino acid sequence was found to be unique to Japanese isolates, and the sequences of
meq are genetically closer to those of Chinese isolates [
22]. To identify the properties of MDV strains distributed in Japan, we previously isolated a field strain of MDV from chickens that showed clinical signs of MD. The isolated strain Kgs-c1 was not contaminated by the three types of vaccine strains [
47]. However, the genetic characteristics of Kgs-c1, except for the
meq gene, is still unknown. Therefore, in this study, to investigate the genetic characteristics of Kgs-c1, we performed whole-genome sequencing. We also compared the sequences with those of MDV strains isolated in other countries.
Discussion
MD is currently well-controlled by vaccination, although it previously caused serious economic losses to the poultry industry. However, the virulence and genetic characteristics of MDV strains have changed over time, and divergence seems to be correlated with the introduction of vaccines [
23,
46]. According to a previous report, the genome sequences of MVD strains are classified into two main clusters, Eurasia and North America [
46]. The genome sequences of MDV strains isolated in USA, China, and Europe have been investigated [
32‐
46]. However, data on the whole genome sequences of MDV strains from other regions are limited. We previously reported changes in the genetic characteristics of
meq genes in Japanese isolates [
22]. In the present study, to compare the genetic characteristics of the whole genome of MDV strains circulating in Japan, we analyzed the whole genome sequence of a Japanese field strain, Kgs-c1, isolated in 2014.
The distinct diversity of the
meq gene has been considered to be correlated with the enhanced virulence of MDV strains [
19,
23]. The
meq gene is thought to be associated with the evolution of MDV virulence, and non-synonymous mutations are frequently observed in
meq genes among MDV strains [
46]. To date, sequences of the
meq genes from MDV strains from various countries have been reported [
5,
24‐
31]. In the present study, the UL–US regions of MDV strains were divided into two clusters, Eurasia and North America, as previously reported [
46], whereas the
meq genes of MDV strains from various regions formed three clusters, North America, other regions including Eurasian countries, and L-
meq. Unfortunately, most of the whole genome sequences of MDV strains for which
meq genes were classified into the L-
meq cluster, in the phylogenetic analysis of the
meq gene, were not available. However, the L-
meq genes of strains 814 and CU-2, which were classified into Eurasia and North America groups, respectively, possess an insertion in the transactivation domain, and therefore, these L-
meq genes seemed to be classified into the L-
meq cluster in this phylogenetic analysis. Thus, the
meq gene and UL–US region seem to indicate similar phylogeny, except for the appearance of the L-
meq cluster. The
meq gene of Kgs-c1, for which the sequence is frequent among Japanese MDV strains, was classified into the cluster of other regions. In addition, the UL–US region of Kgs-c1 was classified into the Eurasian cluster. Taken together, the genetic characteristics of MDV strains distributed in Japan might be largely classified into the Eurasian cluster, similar to that observed with Kgs-c1. However, some
meq genes from Japanese isolates were classified into the North American cluster. Therefore, it is possible that MDV strains with genetic characteristics similar to those of US strains are also present in Japan.
A deletion of amino acid sequences was found in MDV013 (
glycoprotein L)/MDV013.5 (
MHC class II beta chain binding protein) and MDV056 (
probable membrane protein). The deletion in MDV013/MDV013.5 was also found in some highly virulent strains [
19]. However, this deletion does not seem to be correlated with increased virulence [
57,
58]. The Chinese and European strains showed a deletion in the same region of MDV056 [
32,
39‐
46]. However, this deletion was also found in the vaccine strain 814 and therefore, it might not affect virulence, although its correlation with MD pathogenesis is unclear.
The historical background related to the introduction of vaccines in each country is different, and this seems to be correlated with differences in the evolution of MDV genomes in each country [
46]. In Japan, HVT was initially approved for protection from MD in 1972. A few years after the initial introduction of HVT, field outbreaks were sporadically observed in HVT-vaccinated chickens. Therefore, other types of vaccines, specifically CVI988 in 1985 and a bivalent vaccine comprising CVI988 and HVT in 1988, were approved. Thereafter, CVI988 and multivalent vaccines including CVI988 have been widely used to prevent MD occurrences in poultry farms in Japan. In Europe, HPRS-16, which is an MDV strain that was originally isolated in the UK, was initially used as a live-attenuated vaccine [
2]. Later, a vaccine derived from an attenuated MDV strain, CVI988, was used; currently, this vaccine is being used globally [
2]. In contrast, in the USA, HVT was initially developed as a live vaccine [
2]. In addition, a naturally non-pathogenic strain, SB-1, was isolated in the USA in the late 1970s, and has been used as a bivalent vaccine to enhance the vaccine efficacy of HVT [
2]. The historical background related to the use of vaccines between Europe and the USA is thus different, and the background of Japan is closer to that of Europe. Thus, the use of vaccines could induce the evolution of MDV strains in each country, and Japanese strains seemed to develop genetic characteristics similar to those of European strains.
The UL36 protein, a large tegument protein encoded by a member of Herpesviridae, is known to form the innermost layer of the complex protein scaffold between the capsid and envelope [
59]. MDV encodes a ubiquitin-specific protease as part of the N-terminal region of the UL36 protein, similar to that observed in other known herpesviruses [
60]. The UL36 protein was found to be correlated with the tumorigenic activity and replication of MDV via the deubiquitinase activity of the ubiquitin-specific protease [
60,
61]. In contrast, MDV encodes unique repeat sequences at the C-terminal region [
56]. Phylogenetic analysis revealed that the
UL36 gene of Kgs-c1 was classified into the North American cluster, unlike those of the
meq gene and UL–US region. In addition, Kgs-c1 exhibited unique sequences in the coding regions at the 5′ regions of the UL region. Thus, Kgs-c1 might have undergone evolutionary processes different from those of other pathogenic strains, although Kgs-c1 is closer to Chinese and European strains than to US strains in terms of the whole genome sequence.
For the phylogenetic analysis, methods based on the maximum likelihood principle have been often applied, as their accuracy is generally higher than that of other methods. However, these methods require much longer time, and some factors, such as the number of nucleotides, the number of sequences, and the model used for analysis, often affect accuracy [
55]. Therefore, when using these methods, optimization should be considered. In addition, many empirical studies have indicated that other methods, including the minimum evolution principle, provide similar phylogenetic inference by applying the bootstrap test [
55]. A previous study reported that in metagenomic analysis, phylogenetic classification using the novel method (
PhyClass) based on the minimum-evolution principle was as efficient as that with the maximum likelihood methods [
62]. Therefore, in the present study, the genetic characteristics of the Kgs-c1 genome were analyzed using the minimum-evolution method. However, its accuracy might be less than those using the best existing maximum likelihood methods with optimized models. Therefore, other approaches should be applied to analyze the phylogeny of Kgs-c1 more accurately, and assess the biases by the minimum-evolution method.
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