4 DISCUSSION
Marek’s disease (MD) is one of the re-emerging viral diseases of poultry causing serious economic threat to poultry industry (Schat and Nair, 2013). Although vaccines provide strong immunity and highly effective against clinical disease, outbreaks in vaccinated flocks are not uncommon. More virulent and pathogenic MDVs are evolving in recent times worldwide including India. The disease has shifted to visceral lymphomas rather than neural form in recent outbreaks (Kannaki et al., 2020). Moreover, the sub-clinical infection of young chicks with immunosuppressive diseases such as Chicken infectious anemia (CIA), infectious bursal disease (IBD) and fowl adeno virus (FAV) also attribute to the MD outbreaks in vaccinated flocks due to vaccination failure induced by immunosuppression. Some of the unique oncogenes of serotype I MDVs are associated with viral oncogenicity and pathogenicity. In the present study, we investigated the three oncogenes Meq, pp38 and vIL-8 of 17 MDV field strains from MD visceral tumors occurred in vaccinated poultry breeder flocks.
The nucleotide similarity of present MDV strains ranged from 98.5-100% for all three oncogenes due to point mutation at various positions. Although all strains are from same location, the variation may be attributed to high mutation capacity of the field MDVs. Different age groups and genetic background, production type (layer breeder, broiler breeder and native breed), other vaccines and vaccine immunity could be other factors that might have attributed for the emergence of virulent MDV strains.
Meq gene of vv and vv+ MDV pathotypes encode for 339 amino acid containing a N-terminal basic leucine zipper (bZIP) transactivation domain and C-terminal proline rich trans-repression domain. Dimerization and activation of transactivation domain leads to cell transformation and subsequent tumorigenesis (Liu et al., 1999; Levy et al., 2003). Meq of mild or vaccine strains such as CVI988, CU-2 or JM have 59 aa insertion in the C-terminal region known to have suppression effect on the Meq expression and oncogenicity (Shamblin et al., 2004; Chang et al., 2002). Meq of all field MDVs investigated here are of 339 aa length encoded by 1020 bp coding sequence indicating that they are virulent strains. Moreover, the amino acid changes at position 71 and 77, are considered as feature of high virulence MDVs (Shamblin et al., 2004). Mutation at 71 was observed in all current field strains. However, at 77 was not present like Chinese and other field strains (Tian et al., 2011). Mutation at 77 seems to be a feature of virulent strains of USA (Tian et al., 2011). The three amino acid substitutions at positions 119, 153 and 156 unique to very virulent plus strains like 584A and 648A were not present in current strains giving indication that they have not yet evolved as vv+. However, some unique mutations at positions 80 (D80Y), 88 (A88T), 93 (Q93R) and 139 (T139A) were observed in the field strains. Unique amino acid substitutions in Meq gene are recently reported in MDV field strains circulating in Italy (Mescolini et al., 2019), China (Yu et al., 2013), Colombia (Lopez-Osorio et al., 2017) and Turkey (Ozan et al., 2021). Geographically restricted independent evolution of field strains was observed in vaccinated flocks. In the present study also, three unique amino acid substitutions are observed in the field MDV strains. These unique mutations are also observed in few other field strains reported from different regions of this country. Previous studies show the regularity in the mutations of Meqgenes of MDVs from outbreaks of increased virulence (Zhang et al., 2011). Although of GaHV-2 is a DNA virus, Meq gene of GaHV-2 has high mutation frequency of approximately 10-4 substitutions per year like that of RNA viruses (Padhi and Parcells, 2016). High mutation frequency combined with positive selection in vaccinated flocks could drive the emergence of highly virulent pathotypes. However, it needs to be ascertained whether these attribute to a continuing evolutionary drift leading to increased virulence of MDV strains circulating in India.
Number of proline repeats is strongly associated with the level of MDV virulence (Renz et al., 2012; Shamblin et al., 2014). Negative correlation exists between their number and the virulence. The number of four proline repeats ranges between 2-8 in all MDV strains. Generally, virulent to very virulent MDV strains have been shown to possess 5 ‘PPPP’ repeats in Meq gene (Abdallah et al., 2018; Mescolini et al., 2019). All the current strains had 5 repeats. Earlier reported MDV strains from India with virulent to very virulent pathotype also possessed 4-5 ‘PPPP’ repeats (Kalyani et al., 2010; Prathibha et al., 2018). Phylogenetic analysis revealed that current strains clustered with Indian strain from northern part and with very virulent plus ATE 2539 strain from Hungary. Surprisingly, the current strains clustered away from earlier very virulent strains from southern India reported a decade ago (TNN1 & TNN2).
The pp38 gene encodes a 38 kDa phosphoprotein that has a role in cell transformation and virus reactivation from latency (Gimeno et al., 2005). Minor variations were observed in amino acid substitutions among the field strains. Glutamine at position 107 is conserved among virulent MDVs except for CVI988 vaccine strain wherein it has arginine instead of glutamine. The mutation at 107 is considered a reliable biomarker for differentiating field strains from CVI988 vaccine (Baigent et al., 2016). Glutamate at position 109 present in very virulent and vv+ strains was observed in 5 out of 11 sequences investigated. Both these positions viz. 107 & 109 can be considered as virulence indicators as observed earlier in the virulent strains of USA and China (Shamblin et al., 2004; Tian et al., 2011).
The vIL-8 gene consists of 3 exons and encodes 134 aa length peptide that is expressed during cytolytic infection and shows closest homology to mammalian and avian IL-8 (Cui et al., 2004). vIL-8 is considered important for switch of infection from B to T lymphocytes (Schat and Xing, 2000). This gene was shown to be highly conserved among different pathotypes, hence change in virulence can be strongly speculated with any mutations within the gene. The two-point mutations at positions 4 (L4S) and 31 (D31G), initially considered as unique to Chinese MDV strains (Tian et al., 2011) are observed in the current study as well. These mutations were observed in other field strains from Japan and India (Abd-Ellatieff et al., 2018; Prathibha et al., 2018). The phylogenetic tree of the vIL-8 gene revealed the clustering of present strains with vv and vv+ strains from USA and China indicating the virulence of present strains.
Our present study supports the argument that the vaccination pressure imposed the genetic drift of emerging strains towards virulence (Wozniakowski and Salamonowicz, 2014; Padhi and Parcell, 2016; Nair, 2018). Sequence and phylogenetic analyses of oncogenes from field MDVs from vaccinated flock indicate these strains possess molecular features of very virulent strains. Although vv+ strains are not yet reported in Indian poultry flocks and not observed in the current investigation, the rate of evolution of Meq gene in recent times by various reports suggest the rapid evolution in MDV strains towards severe pathogenicity. Meq gene followed by vIL-8 and pp38 genes could be of additional value in deciphering the pathotype of MDV strains. Molecular characterization of oncogenes would be the most rapid, reliable and affordable method to suggest the virulence of field strain instead of tedious in vivopathotyping assays. In conclusion, continuous surveillance is mandatory to monitor the emergence of virulent MDV strains and to devise appropriate vaccination strategy and bio-security programs.