Molecular Method of Marek’s Disease Virus Serotype 2 Detection using Polymerase Chain Reaction in Chicken

Marek’s disease virus is an alpha herpesvirus belonging to the genus Mardi virus produces malignant lymphomas in chicken few weeks after infection (Calnek and Witter, 1997; OIE, 2017) Marek’s disease virus is under herpesvirus group having three serotypes namely Gallid herpes virus II (serotype1 and also known as Mareks disease virus), Gallid herpesvirus III (serotype2) and Meleagrid herpes virus I or herpes virus of Turkey bird (HVT) (serotype 3). Serotypes 2, 3 include all the naturally avirulent strains, some of which are used as vaccines. Antigenically related HVT strains are also used as a vaccine against MD (OIE, 2017).
       
The MDV infection is detected in inoculated susceptible cells and the cultures are incubated to find the viral plaque appearance and also by immunofluorescent assay (Lee et al., 1983).
       
At present the diagnostic techniques to detect MDV 1 and 3 are by polymerase chain reaction (Becker et al., 1992; Silva, 1992, Zhu et al., 1992, Bumstead et al., 1997; Handberg et al., 2001, Sadeghi et al., 2006) in suspected field samples. The vaccination against Marek’s disease virus is currently available by using serotype 2 strain SB 1 or HVT or a mixture of both (Witter and Lee, 1984). In Asia, the serotypes are used as a vaccine candidate against Marek’s disease such as serotypes 2 and 3 (SB1 and HVT) except in Japan, Europe, USA, South America using cv1988+HVT strains (Baigent et al., 2006). Both ALV subgroup E (ALV-E) and MD Serotype 2 are having an important role in the enhancement of spontaneous Lymphoid Leucosis-like tumors in susceptible chickens (Mays et al., 2019). In ovo vaccination using MD serotype 2 augment the incidence of spontaneous Lymphoid Leucosis-like lymphomas in transgenic chicken line (ALVA6) chickens (Cao et al., 2014). In this regard, separate development of polymerase chain reaction for serotype 2 is very much needed in India by using unique region of MDV serotype 2 with the designed primers to amplify the Long Open Reading Frame (LORF) gene of MDV serotype 2 SB1 strain.

Vaccine purchase
 
The commercially available serotype 2 vaccines (SB1+HVT combined bivalent) and serotype 3 vaccine (HVT only monovalent), were purchased from Zoetis Company, USA and utilized in this study.
 
MDV and other strains
 
The field MDV suspected birds feather follicles, liver and spleen were collected from Namakkal District, Tamil Nadu andhra Pradesh, Maharashtra and Puducherry, India. Avian Leucosis Virus and Reticuloendotheliosis Virus were collected from Animal Biotechnology Department, Madras Veterinary College, Chennai.
 
Isolation of DNA
 
The DNA was extracted using a DNA extraction kit (QIAGEN, USA) from the cell-associated vaccine (serotype 2/3), lyophilized vaccines (serotype 3) as per the protocol given in the kit. The DNA was isolated from MDV infected chicken feather follicles, liver and spleen by phenol-chloroform DNA isolation method (Handberg et al., 2001). The purity and concentration of DNA measured using Nanodrop one (Thermo fisher scientific company, USA) and stored at-20°C.
 
MDV serotype I specific PCR
 
Chicken feather follicles, liver and spleen were screened for MDV serotype I using previously reported primers (Sathish et al., 2012).
 
Primer designing for PCR
 
Serotype-II specific primers were designed using primer3.0 software (frodo.wi.mitedu/primer3/input.htm) targeting the LORF gene (Table 1) which is specific to the serotype 2 SB 1 strain.
 

 
PCR optimization
 
DNAs isolated from vaccines were used as templates for optimizing the PCR parameters such as melting temperature (Tm value) from 53°C to 61°C and primer concentration (0.1pmol/µl to 10 pmol/µl.)
 
PCR conditions
 
DNA from vaccines were subjected to PCR with the LORF primers followed by the reaction conditions as initial denaturation at 94°C for 1 min, 35 cycles of denaturation at 94°C for 1min, annealing at 59°C for 1min and extension at 72°C for 1 min and final extension at 72°C for10 mins. PCR products were checked in 1.5% agarose gel electrophoresis.
 
Specificity of primers
 
LORF gene-specific primers of SB-1 strain were cross-checked with HVT and MDV-1strains and the other oncogenic viruses of ALV and REV under the same PCR conditions.
 
Sensitivity of primers
 
The PCR was carried out with different DNA concentrations (0.5 ng/µl to 30 ng/µl) of vaccines to detect the sensitivity of the assay.
 
Sequence analysis
 
The positive PCR products were purified and sequenced by Eurofins Company. The sequences were aligned and showed 99% homology with MDV serotype II by BLAST analysis (Fig 1).
 

Optimization of PCR
 
The DNA template isolated from vaccines for PCR optimization with PCR primers is shown in the amplification of the LORF gene (270 bp) for MDV serotype 2 at an optimized annealing temperature of  53°C (Fig 2) with a Primer concentration of 3 pmol/µl (Fig 3).
 

 

 
Specificity of PCR primers for serotype 2
 
The LORF gene of SB1strains specific primers was cross-checked with   HVT and Serotype I strains and the results revealed that these primers did not cross-react with HVT and Serotype I strains (Fig 4). Further, it was not amplified with other oncogenic viruses ALV and REV (Fig 5).
 

 

 
Sensitivity of primers
 
Sensitivity was analyzed based on the different DNA concentrations and showed amplification up to 0.5 ng/µl for the PCR assay (Fig 6).
 

       
The screening of field samples from various parts of the country revealed that feather follicles, liver and spleen, did not show positivity in the developed PCR assay whereas MDV serotype vaccine SB1 strain samples showed positivity as amplification at 270 bp in 1.5% agarose gel electrophoresis (Table 2).
 

       
Marek’s disease virus is having three serotypes serotype 1, (oncogenic), serotype 2 and 3 (non-oncogenic). The polymerase chain reaction for the MDV detection method is simple and results are detected within a day (Sadeghi et al., 2006). PCR is a direct detection assay of Marek’s disease virus as per the earlier report of Silva (1992). The suspected antigen of Marek’s disease virus is cross-reacting with others serotypes and MDV (Rispens et al., 1972; Hirai et al., 1986). Sadeghi et al., (2006) developed a polymerase chain reaction for detection and differentiation of MDV 1 and HVT strain. Sathish et al., (2012) reported the multiplex PCR assay for the detection of different avian oncogenic viruses (MDV, ALV, REV).
       
A perusal of literature, the MDV serotype 2 is having the property to augment the incidence of spontaneous Lymphoid Leucosis-like lymphomas in commercial chickens (Mays et al., 2019) and transgenic chicken line (ALVA6) chickens (Cao et al., 2014). Serotype 2 specific PCR is not available to diagnose MDV serotype 2 in India and this study has given the information of specific PCR of serotype 2 with amplification at 270 bp size which is unique to the LORF gene of MDV serotype 2 SB1 strain. The forward primer and reverse primer pair used in the study were given amplification at 270 bp that encodes the LORF gene of SB1.
       
The developed assay for detection of serotype 2 primers as given specific amplification of the LORF gene of serotype 2 SB1 strain without any cross-reaction with another serotype of MDV 1 and 3 indicated that specificity to serotype 2 only. Screening of field samples of 260 along with three MDV serotype 2 vaccine SB1 strain samples showed that no positivity was observed in the 260 filed samples but in the vaccine strain samples of MDV serotype 2 vaccine SB1 showed positivity by amplification at 270 bp. In the present study, the specific PCR for serotype 2 of MDV give an idea about disease outbreaks from the screened field samples that did not occur due to MDV serotype 2 and it could be occurred due to vaccine failure or virulence strains of serotype 1. In addition to that, the study indicated that MDV serotype 2 SB1 strain did not cause an outbreak in chicken and might be informed that there is no incidence of spontaneous Lymphoid Leucosis-like lymphomas with augmentation property of MDV serotype 2 (Cao et al., 2014; Mays et al., 2019). 
       
The PCR for serotype 2 MDV detection is useful to find the Marek’s disease virus caused by MDV 2 or not. This assay could be employed to differentiate Marek’s disease bivalent vaccine (SB1/HVT) from the monovalent vaccine (HVT). This assay is useful to detect Marek’s disease caused by serotype 2, which is commonly used as a vaccine strain of MDV.

In the present study, MDV serotype 2 specific PCR was developed and screening of field suspected MDV samples did not show positivity to MDV serotype 2 while vaccine samples showed positivity for MDV serotype 2.

The author is greatly acknowledging the Department of Biotechnology (DBT), Government of India, providing financial support to carry out this research work.

The authors declare no conflict of interest.