Molecular analysis of oncogenicity associated gene “vIL8” of serotype 1 Marek’s disease virus isolates from India

Marek’s disease (MD) is one of the most common lymphoproliferative diseases of chicken resulting in mononuclear cell infiltration of one or more of the visceral organs and nerves (Witter and Schat, 2003). Namakkal is the second largest egg bowl in India where the intensity of poultry rearing keeps on increasing which ushers in reemergence of MD. Marek’s disease has a tremendous economic impact because of continuing losses due to the disease and also due to cost of vaccination.
 
Marek’s Disease Virus is a cell associated herpesvirus. The genome of all three MDV serotypes is broadly similar in their sequences. It consists of a linear, double standard DNA of 160 – 180 kbp in size. The genome consists of unique long (UL) and unique short (US) regions. Several genes unique for MDVs have been identified (Witter and Schat, 2003).
 
There is continuous evolution that are taking place in MDV towards greater virulence (Suresh et al., 2017), which has resulted in increased losses from MD in vaccinated flocks now a days (Suresh et al., 2015). A thorough understanding of the genes involved in replication, immune modulation, and oncogenesis holds the key to the improved vaccination strategies, based on targeted mutations in specific oncogene and oncogenecity associated genes present in MDV genome.
               
The vIL-8 gene is involved in early cytolytic infections in lymphoid organs, because of the impaired cytolytic infections, the overall transformation efficiency of the vIL-8 deleted mutant is much lower, as reflected by the reduced number of transformed cells at 5 weeks post-inoculation and the presence of fewer gross tumors (Cui et al., 2004). Therefore, isolation of field MDVs and sequence analysis of vIL8 gene can help us to decipher the molecular characters of the isolates involved in recent epidemics in India. In recent years, isolation and sequence analysis of MDVs in India were reported in breeder or layer flocks which had been vaccinated with HVT or bivalent vaccine and confirmed that virulent and very virulent MDVs are circulating in India (Raja et al., 2009; Suresh et al., 2013). However, sequence analysis data for vIL8 gene is not available in India although recent sequence data for Meq and pp38 genes are published. Hence, molecular characterization of vIL8 gene for the first time in India would be of use to correlate the results of this study with available data pertaining to other critical genes including Meq and pp38 to aid in selection of better vaccine candidate. Based on the above said facts this st udy was planned to analyze the nucleotide sequences of oncogenecity associated vIL8 gene of the three field Indian isolates.

Isolation of Serotype 1 MDV
 
Samples: Eighty six blood samples were collected from 15 commercial layer and broiler breeder farms throughout Tamilnadu and parts of Karnataka state of India in which MD outbreak occurred in spite of vaccination with monovalent and bivalent vaccines. EDTA was added to blood samples and samples collected from individual farm were pooled to represent that particular farm.
 
Duck Embryo Fibroblast Culture (DEF): Primary cell cultures were prepared from the 10 to 12 day-old embryo- nated duck eggs as per the protocol of supplemental assay methods (2005), Centre for Veterinary Biologicals, USDA.
 
Co-cultivation of Lymphocyte and DEF cells: Lympho- cytes were collected by using ficoll-paque aseptically from the blood samples which were 132 bp repeats positive (Two or three copies) and co-cultivated with DEF cells (Tian et al., 2011). The co-cultivated monolayers were observed every day for five to seven days. After three blind passages, the presence of serotype 1 MDV in DEF was verified by PCR detection of 132 bp repeated sequence.
 
Infecting the Chicken Embryo Fibroblast (CEF) monolayer: The DEF harvests which were positive for two or three copies of 132 bp repeats having no contamination of Avian Leukosis virus (ALV) and Reticuloendotheliosis virus (REV) screened by multiplex PCR kit (Gopal et al., 2012). The harvests free from ALV and REV were further passaged in CEF monolayer until the appearance of typical MDV plaques. The infected monolayers were observed every day for the formation of plaques upto 7 days.
 
Amplification of vIL8 gene
 
DNA extraction: Total DNA was extracted from CEF cells using EZ – Spin Column Viral DNA Mini – Preps Kit (Bio Basic Inc. Canada) and stored at -20ºC.
 
Polymerization chain reaction: Primers for oncogenicity associated vIL8 gene (Tian et al., 2011) were synthesized at Bioserve Pvt. Ltd., Hyderabad and used for PCR amplification.
 
The primer sequences are shown in Table 1.
 

 
PCR was carried out in a final reaction volume of 50 μl using 200 μl capacity thin wall PCR tube. A reaction mixture constitutes Red Dye Master mix (2x) 25 µl, Forward and Reverse Primers 2 µl each (10 pmol/μl concentration), Template DNA 2 µl (50ng/µl) and Nuclease free water 19 µl. The PCR protocol was initial denaturation at 94ºC for 4 min., denaturation at 94ºC for 1 min., annealing at 56ºC for 1 min., extension at 72ºC for 1.5 min. and a final extension at 72ºC for 10 min. for 35 cycles.
 
Gel Electorphoresis: To confirm the targeted PCR amplification, five μl of the PCR product from each tube was loaded and electrophoresed along with 1200bp DNA molecular weight marker (Bio Rad, USA) on 1.5 per cent agarose gel containing ethidium bromide (at the rate of 0.5μg/ml) at constant 80V for 45 min in 0.5X TAE buffer. The amplified product was visualized as a single compact band of expected size under UV light and documented by gel documentation system (BioRad, USA).
 
Sequencing and analysis: It was done by automated sequencer at Scigenome laboratories, Cochin. The obtained nucleotide sequences and deduced amino acid sequences of oncogenic vIL8 gene of three MDV isolates were edited using the Editseq programme in the Lasergene package (DNASTAR Inc, Madison, WI, USA), and compared with other reference MDVs for the homology analysis with the use of MegAlign programme in the same package. Phylogenetic analysis was performed with the neighbor-joining method using MEGA version 4.0. The bootstrap values were determined from 1000 replicates of the original data. Six reference strains were chosen for comparison of vIL8 genes and these reference strains were retrieved from the GenBank database, and the backgrounds of the reference strains used in this study are listed in Table 2.
 

Isolation
 
Five representative MDV serotype 1 strains were isolated from 5 of 15 farms by using DEFs culturing (33.33%). Three out of five MDV isolates were free of ALV and REV. They were adapted to CEFs and produced typical cytopathic effect (CPE) (MDV serotype 1 Plaques) after 2-4 passages (Fig 1). The MDV isolates of two farms were discarded because of concurrent infection with ALV or REV. The isolates were named as Ind/ TN/11/01, Ind/KA/12/02 and Ind/ TN/12/03 respectively.
 

 
Amplification of oncogene vIL8
 
vIL8 gene of all the three isolates was amplified by PCR conditions given in method. All three isolates produced amplicon of size 887 bp in agarose gel (Fig 2).
 

 
Sequencing of vIL8 gene
 
The sequences of the vIL8 genes of the three MDV isolates of this study were submitted to GenBank and the details are given in Table 3.
 

 
Homology analysis of vIL8 genes between isolates and reference strains
 
Homology analysis of vIL8 gene had revealed 99.8-100 per cent homology of nucleotide sequences among the three isolates. The isolates were shown to have a homology of 94.6-95.1 per cent with various isolates of China and 96.6 – 99.5 per cent with other isolates of USA as shown in Fig 3. However there were no earlier sequencing reports of vIL8 gene in Genbank from India.
 

 
Alignment analysis of vIL8 genes between isolates and reference strains
 
Alignment analysis of vIL8 complete nucleotide sequences of the three field isolates and six published MDVs were performed. Nucleotide mutations were observed when RB1B strain was used as reference strain. The nucleotide mutation in the vIL8 gene of MDVs had displayed regularity at three positions, including 112 (T to C), 329 (T to C) and 368 (A to G) and occurred in entire field MDV isolates of this study. At position 265 the very virulent plus strains including 584A, 648 A and very virulent strain Md5 possess a mutation G to V C (Fig 4). There are changes noted in amino acids in entire isolates, at site 4 (L to S) and at site 31 (D to G) (Fig 5).
 

 

 
Phylogenetic analysis of vIL8
 
Phylogenetic analysis on the vIL8 sequence of three isolates and other six reference strains showed that the analyzed nine MDVs could be separated two groups (cluster 1 and cluster 2) (Fig 6).
 

 
Marek’s Disease Virus is a member of the genus Mardivirus that consists of serotypes 1 and 2 (MDV-1 and MDV-2) as well as serotype 3 or herpesvirus of turkeys (HVT) (Davison, 2002). Among them, the only serotype 1 is oncogenic, and some of the unique genes such as Meq (Jones et al., 1992), pp38 (Cui et al., 1991), vIL8 (Parcells et al., 2001) were reported to be associated with viral oncogenicity and pathogencity. In this study, homology comparison of the nucleotide of vIL8 of three isolates and six other reference strains were conducted.
 
The vIL8 gene located in the long repeat region and was originally identified as a spliced Meq variant (Peng and Shirazi, 1996). The gene consists of three exons and is expressed during cytolytic infection and encodes for 134 aa long chemokine that had high homology to human and chicken cellular IL-8 (Liu et al., 1999; Parcells et al., 2001). However, it is not essential for viral replication, latency but for tumor formation and virus transmission (Cui et al., 2004).
 
Even though on one hand Cui et al., (2004) claims no role of vIL8 gene in latency reactivation and replication, on the other hand accepts the fact that the impaired early cytolytic infection due to the deletion of vIL8 leads to weak activation of T cells, resulting in reduced numbers of target cells for transformation and significantly decreased pathogenicity and tumor incidence. This proves the fact that vIL8 is a chemokine and even though not directly involved in oncogenesis but acts as an oncogenecity associated factor. Any mutation in vIL8 will have a direct impact on clinical signs by the way of increased occurrence of tumors. 
 
Nucleotide mutations were observed when RB1B strain was used as reference strain in alignment analysis of vIL8 complete nucleotide sequences of the three field isolates. The nucleotide mutation in the vIL8 gene of RB1B displayed regularity at three positions, including 112 (T to C), 329 (T to C) and 368 (A to G) which was also noticed in entire field MDV isolates of this study. At position 265 the very virulent plus strains including 584A, 648 A and very virulent strain Md5 possess a mutation G to C. This is not found in RB1B and isolates of this study. This indicates that isolates of present study are closer to very virulent RB1B than very virulent plus strains including 584A, 648 A and very virulent strain Md5.
 
Zhang and Qin  (2003) described that vIL8 was very conservative in 648A, RB1B, MD11 and GA strains, but Tian et al., (2011) could find two special point mutations at position 4 (leucine to serine) and 31 (aspartate to glycine) in the field MDVs from China. They speculated that the point mutations at position 4 and 31 could be considered as the features of field MDVs in China.
 
A local strain of China SD2012-1 had the amino acid mutation of vIL8 gene at position 4 (L to S) and position 31 (D to G), which displayed regularity of strains isolated from China (Gong et al., 2013). In this study nucleotide mutation in the vIL8 gene of MDVs displayed regularity at three positions, including 112 (T to C), 329 (T to C) and 368 (A to G) occurred in entire field MDV isolates of this study. This could be the feature of virulence of this local isolates. The deduced amino acid sequences of the isolates of this study with respect to vIL8  chemokine protein  had also shown similar changes like Chinese isolates. At position 265 the very virulent plus strains including 584A, 648 A and very virulent strain Md5 possess a mutation G to C which is different from local field isolates.
 
Incidence of lymphoma was very high (86%) in Ind/TN/12/03 inoculated group of birds during experimental study followed by Ind/KA/12/02 (66%) and Ind/TN/11/01 (50%) inoculated groups as observed by the author in earlier study (Suresh et al., 2015). This heightened pathogenecity may be due to change that had occurred at molecular level and subsequent change in chemokine protein (vIL8) which plays a crucial role in lymphoma formation as stated by Cui et al., (2004).
 
Phylogenetic analysis on the vIL8 sequence of three isolates and other six reference strains showed that the analyzed nine MDVs could be separated two groups (cluster 1 and cluster 2). Cluster 1 contains very virulent plus strain 584 A, 648A, very virulent strains Md5, RB1B and TQ2. Cluster 2 contains all the field isolates of this study. This is suggestive of independent evolution of the local isolates.
 
The molecular analysis had shown mutation in vIL8 gene of MDVs isolated from southern part of India with regularity at three positions, including 112 (T to C), 329 (T to C) and 368 (A to G). At position 265 the very virulent plus strains including 584A, 648 A and very virulent strain Md5 possess a mutation G to C. This is not found in RB1B and isolates of this study. This indicates that isolates of present study are closer to very virulent RB1B than very virulent plus strains including 584A, 648 A and very virulent strain Md5.

The very virulent nature of the isolates at molecular level warrants the adaptation of these isolates in suitable cells and subsequent pathotyping assay to explore the possibility of using them as vaccine candidate. The authors are highly thankful to Tamilnadu Veterinary and Animal Sciences University (TANUVAS), Chennai for necessary funding and The Dean, Veterinary College and Research Institute, Namakkal for providing facilities.