Introduction
Marek's disease (MD) is a highly contagious immunosuppressive disease that is characterized by paralysis and lymphoma development of T-cells in viscera and muscles [
1]. The etiological agent is Marek's disease virus (MDV), or gallid herpesvirus 2 (GaHV-2), a member of the genus
Alphaherpesvirus, family
Herpesviridae, that has an enormous impact on the poultry industry [
2]. The molecular basis for increased MDV virulence is currently unknown, but mutations in some regions of the viral genome, including the
Meq (Marek's disease virus EcoRI fragment Q)
, phosphoprotein 38 (
pp38)
, viral interleukin-8 (
vIL-8)
, UL1, and
UL44 genes have been implicated as virulence factors [
3‐
7].
The MDV genome consists of a double-stranded linear DNA molecule of about 180 kbp. It consists of several regions, namely, long unique (UL) and unique short (US) regions flanked by long terminal repeats (TRLs) and short terminal repeats (TRSs), long internal repeats (IRLs) and short internal repeats (IRSs). The genome structure and the gene content of each region are similar in all GaHV-2 serotypes, but there are key differences [
8]. Oncogenic serotype 1 is characterized by the presence of the
Meq oncogene and other unique genes, including
pp38, vIL-8, and
vTR, in the repeat regions, particularly the TRL region. The
Meq gene is considered the major factor responsible for tumorigenesis in chickens, since its deletion leads to a breakdown of T-cell transformation [
9]. In addition to the
Meq gene, which is directly responsible for transformation, the major lytic antigen pp38 has been reported to be associated with enhanced virulence and is highly expressed during lytic infection and lymphoma formation [
10,
11]. There are other genes, such as
vIL-8, whose deletion or mutation may decrease tumor development and lead to attenuation of virulence [
5,
12]. Another gene that is suspected to contribute to the virulence of some hypervirulent MDV strains is the
UL1 gene, which codes for glycoprotein L (gL) [
6]. This protein forms a hetero-oligomeric complex with glycoprotein H (gH-L) that plays an important role in entry of the virus into host cells and cell-to-cell infection [
13] An MDV-specific cytotoxic immune response is induced by the MDV gC protein and, to a lesser extent, the gL protein [
14]. The gC protein, which is encoded by the
UL44 gene, appears to be involved in virus-host interactions and is important for attenuation of virulence [
7].
Due to mutations in the viral genome, the severity of disease varies among GaHV-2 isolates, leading to ever-changing pathotypes that overwhelm vaccine protection, and efforts have been made to identify new emerging pathotypes in order to ensure vaccine protection [
15]. Sporadic epidemics of MD have been reported worldwide, even in vaccinated flocks [
16], indicating that the virulence of MDV has increased in recent decades, and some of the more recent isolates are more pathogenic to chickens than older isolates [
17,
18]. Thus, the increased use of vaccines has led the virus to evolve toward higher virulence, which can overcome the protection conferred by currently available vaccines [
19]. It has been suggested that the widespread use of the CVI988 vaccine strain may have led to the emergence of new, more virulent pathotypes [
20], and there are serious concerns that such pathotypes are circulating in Tunisia, especially due to the excessive use of vaccines. This poses a threat to the poultry industry and gives a higher priority to research, especially in the absence of relevant studies, particularly in Tunisia. Therefore, the main objective of this study was to determine the pathotypes of circulating GaHV-2 strains on Tunisian farms based on analysis of the
Meq,
pp38,
UL1,
UL44, and
v-IL8 genes of two MDV field isolates. This will provide a basis for the development of more effective vaccines.
Discussion
In recent years, numerous isolations of vv and vv + MDV strains from vaccinated chickens have been reported, and very common forms of proliferative lymphatic diseases have been described [
20]. The virulence of MDV isolates has increased in recent decades, and some recently isolated vv and vv + strains have been shown to be more pathogenic for chickens than the older ones [
28]. Recombination events among MDV strains could be among the mechanisms by which virulence increases [
29]. It is therefore justified to be concerned about new emerging strains that can break vaccine protection.
Some GaHV-2-specific genes, including
Meq, pp38, UL1, UL44 and
vIL-8, contain sequence differences that are associated with oncogenicity, viral pathogenicity and virulence [
26,
30]. In this study, to better understand the evolution of Tunisian isolates, we studied the pathotypes of MDV strains circulating on farms based on the
Meq, vIL-8, pp38, UL1 and
UL44 genes.
The
Meq gene has been studied extensively and appears to correlate with virulence [
3]. Indeed, it has been reported that a change of serine to alanine at position 71 is a characteristic of hypervirulent strains of MDV [
3]. This has been observed in the Chinese vv isolate SD2012-1 (KC511815) and vv + isolate JZ2014 (KP144355), the American isolates N (AY362718), W (AY362723), 584A (DQ534532), L (AY362717) and X (AY362724), and the Tunisian isolates TN1013/16 (MK041219) and TN1014/16 (KY113150). However, some vv strains, such as 02 LAR (EF523772) and Woodlands1 (EF523775), have a serine at this position, casting doubt on whether the presence of this mutation is a reliable criterion for classifying MDV pathotypes.
The substitution of glutamate by leucine at position 77 has been reported by Shamblin et al. [
3]
. to be a feature of hypervirulent strains. However, isolate JZ2014 (KP144355), the Colombian isolates UDEACO04 (KU058701) and SD2012-1 (KC511815), and the Tunisian isolatesTN1013/16 (MK041219) and TN1014/16 (KY113150), have glutamic acid at position 77 but are classified as vv and vv + strains. This suggests that the presence of glutamic acid at position 77 is not necessarily a characteristic of low-virulence MDV strains. On the other hand, the presence of the amino acids R
119, Q
153 and A
176, has been shown to be a common feature of the American vv isolates 549 (AY362714) and 595 (AY362715) and the American vv + isolates N, L, X, 584A, and 684A (AY362725), in contrast to a report by Tian et al
. [
4], who suggested that these mutations are unique in vv + strains. However, the Tunisian isolates share R
119, and P
176 as well as P
153.
MDV strains with high virulence have been shown to contain mutations at the second position of the proline-rich region (PRR): PPPP > P (Q / A / R) PP [
3]. Attenuated strains have more PPPP motifs, whereas most virulent strains have more interrupted motifs. Analysis of the Meq protein amino acid sequences of theTN1013/16 and TN1014/16 isolates revealed a point mutation in the proline-rich region at position 217, interrupting the PPPP motif. A variable number of PPPP motifs in the central PRR of the Meq protein was observed in low-virulence strains, and in fact, eight repeats were detected in the vaccine strain CVI988, and nine and 10 in the Italian strains GaHV-2/Italy/Ck/847/15 and GaHV-2/Italy/Ck/847/17, respectively [
31]. In contrast, hypervirulent strains have low number of these motifs, and the smallest number was observed in the vv + strain 648A, with only two motifs. The hypervirulent vv and vv + strains contained interruptions in the proline repeats at the second position (P > Q
153, P > A
176 and P > A
276), and the largest number of such interruptions was observed in the most virulent strains [
32]. Both TN1013/16 and TN1014/16 contained only the interruption at position 217 that is present in all virulent strains except RB1B.
On the other hand, Wajid et al. [
33]
. have suggested a correlation between the proline content and the virulence of emerging strains. Analysis of reference strains showed that this parameter is inversely proportional to virulence. In this case, this percentage is around 21.18% in the Tunisian isolates, reinforcing the hypothesis that they are very virulent.
Mutations at positions 110 and 271 appear to be characteristic of Tunisian and Italian isolates [
31]. Such mutations and their overall sequence similarity might suggest that migratory birds such as ducks and white footed geese play a role in transporting the virus from one country to another [
34]. The genetic divergence of MDV strains in the
Meq gene is more obvious than in the
vIL-8, pp38, UL1 and
UL44 genes, making the
Meq gene most appropriate for phylogenetic analysis.
In addition, the
vIL-8 gene, located in the long repeat (RL) region, was initially identified as a spliced variant of the
Meq gene [
35]. It is highly conserved in all strains but with some variability at sites 4 and 31 of the vIL-8 protein. However, these mutations are not characteristic of virulent strains.
As reported by Shamblin et al
. [
3], we did find some mutations in the
UL1 and
UL44 genes that strictly correlate with the virulence level. These mutations map to the putative signal cleavage site of the
UL1 genes, and are found in four out of 11 vv + MDVs, but also in one vvMDV (643P), indicating that it does not correlate with enhanced virulence.
Although several early genes have been shown to be essential for viral replication, and thus for initiating the transformation pathway and subsequent development of clinical signs of MD, the
Meq gene is considered to be the primary oncogene of MDV, with other genes serving as auxiliary factors [
10]. Similarly, although the
Meq gene is mainly expressed during the latent state, studies have indicated that the
Meq gene can also be expressed early in infection [
10], suggesting that variations in the
Meq gene sequence can have a significant influence on virulence. However, previous gene sequencing studies have not revealed mutations in other genes that are consistently correlated with virulence [
3].
Our findings showed that there is significant polymorphism in the MDV Meq gene, which is a key gene in the induction of lymphoid tumors. The Tunisian isolates had two point mutations in the Meq gene that might be associated with virulence. In addition, the overall proline content and pattern of PPPP repeats showed strong correlation with the virulent pathotype. Although the virulence of a specific isolate is unlikely to be determined solely by mutations in a single gene, the results of our work suggest that Meq gene sequencing and protein analysis can provide a useful indication of the virulence of these isolates. However, confirmation of the pathotype requires in vivo experiments.
The CVI988 vaccine is widely used around the world. In China, it has been noted that immune failure tends to occur in only a few chickens. A number of vv strains have been isolated from different parts of China [
36], raising the question whether the currently available vaccines are able to protect chickens against these highly virulent strains.
The weak protective effect of MDV vaccines has led to MD outbreaks [
16], and the emergence of MDV strains with increasing virulence is therefore an important issue for the poultry industry.
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