Adaptation of wild strain of duck plague virus in cell culture systems

Duck rearing is a profitable poultry industry because of its multiple out-put in the form of egg, meat and feather (Rajput et al., 2014)  India has a duck population of 23.539 million constituting 3% of total poultry population (GOI, 2012) and concentrated in the states of Assam, West Bengal, Andhra Pradesh, Tamil Nadu, Uttar Pradesh, Bihar and Orissa (Narhari, 2009).
        
Assam ranks as highest duck populous state of the country having 7.31 million ducks. However, population of duck as a whole has decreased at a rate of 14.85 per cent during the period 2007 to 2012. One of the main reasons of declining duck population may be attributed to frequent occurrence of infectious diseases like duck plague and duck cholera (Neher et al.,2018).
        
The duck virus enteritis (DVE) also known as duck plague is an acute contagious herpes virus infection of duck and other domestic and wild waterfowls (Leibovitz, 1991). As per the recent taxonomic classification by ICTV, duck plague virus (DPV) has been classified into the genus Mardivirus, subfamily Alpha-herpesvirinae of the family Herpesviridae (King et al., 2012 Fadly et al., 2008; Li et al., 2009). The disease is known to have global distribution and due to high mortality, decreased egg production and hatchability, significant economic losses are associated with DPV across the world (Wang et al., 2013).
        
The disease was first reported by Baudet in Netherlands (Baudet, 1923). After that the disease was subsequently reported in many part of the world. In India, DVE was first diagnosed from a severe outbreak in the state of West Bengal during early nineteen sixties (Mukherjee et al., 1963a, 1963b). Thereafter, the disease has spread to different parts of the country and frequent outbreaks have been recorded in the duck-rearing areas including Kerala (Kulkarni et al., 1995), Tamil Nadu (Chellapandian et al., 2005), West Bengal (Bhowmik and Chakrabarty, 1985) and Assam (Chakrabarty et al., 1980).
      
The disease has caused much havoc in Assam. Several studies have been carried out to study the virus epidemiology, pathology, diagnostics and vaccine development. Occurrence of the disease is associated with heavy mortality in domestic and wild ducks (Dhama et al., 2017). In a recent study it was reported that as many as 8 districts of Assam were found to be affected with DPV infection causing heavy mortality of 81.01% ( Neher et al., 2018).
        
Vaccination is the only option for the prevention and control of Duck plague. In India, vaccination is done with chicken embryo adapted live virus vaccine. This vaccine is not free from egg borne infection, influence of maternal antibody and involves high production cost (Kulkarni et al., 1998; Ferguson et al., 2005; Mondal et al., 2010). In this present situation, a safe, potent and cheap DPV vaccine is required for controlling the menace of Duck plague. The vaccine must be easy to produce in large scale and free from other adventitious infectious agents. The use of chicken embryo fibroblast and a certified cell line may, probably, be the best option to achieve this. Although, work is being done on adaptation of vaccine strain of DEV in various primary cell cultures, very few reports are available on the adaptation of the local strain of duck plague virus in CEF and certified cell lines. Furthermore, studies are required to understand the potential of a locally isolated DPV as a vaccine candidate. This paper describes about adaptation of wild strain of DPV in primary cell culture as well as in established cell line.

Source of wild strain of duck plague virus
 
The virulent strain of Duck Plague Virus available in the virus repository of the department of Microbiology (DP/AS-Km/0019) was used to adapt in cell culture. The virus was revived in 1-2 months old maternal antibody free ducklings. Internal organs like liver, spleen, kidney, oesophagus, annular ring and heart were collected and samples were processed for isolation in duck embryo fibroblast (DEF) primary cell culture which was used for adaptation in chicken embryo fibroblast (CEF) as well as in cell lines.
 
Adaptation of DPV in CEF
 
The primary chicken embryo fibroblast cell culture was prepared from 9-11 day old embryonated chicken eggs as per standard protocol (Rai, 2010). The CEF monolayer was grown in tissue culture flask (25cm²) at 37°C under 5% CO₂ in Eagle’s Minimum Essential Medium (EMEM) containing 10% foetal bovine serum (FBS, Hyclone) supplemented with 2mM L-Glutamine and antibiotics at the rate of penicillin 100 units, streptomycin 100 μg and amphotericin B 25 μg per ml. A local strain isolated in DEF was used for propagation in chicken embryo fibroblast. The lyophilized virus (DP/AS-Km/0019) of DPV) was reconstituted in Hank’s balanced salt solution (HBSS) and treated with cocktail antibiotic solution (Sigma 100X, USA) @10µl /ml of inoculums. The monolayer was thoroughly washed and inoculated with 1 ml virus inoculums per 25cm² cell culture flask (Nunc, Denmark) and kept at 37°C for 1 hour to facilitate proper adsorption of virus on to the cells. The un-adsorbed viruses were washed with Ca²⁺ Mg²⁺ free PBS. Then, maintenance medium containing 5 percent foetal bovine serum was added to the flask and incubated at 37°C for 3-4 days. Un-inoculated healthy monolayer was kept as control. The cell monolayer was observed daily under inverted microscope for any change of cell morphology. The virus was harvested after appearance of cytopathatic effect (CPE) and was stored at -30°C for future use.
 
Adaptation of DPV in Vero cell line and QT-35 Cell line
 
The Vero and QT-35 cell lines were maintained in the Department of Microbiology, College of Veterinary Science, AAU, Khanapara using Eagle’s Minimum Essential Medium (EMEM) containing 10% foetal bovine serum (FBS) supplemented with 2 mM L-Glutamine. Antibiotics were added to the growth medium at the rate of penicillin 100 units, streptomycin 100 μg and amphotericin B 25 μg per ml. The cells were grown in 25 cm² cell culture flask (Nunc, Denmark) and the flasks containing confluent monolayer were used for adaptation of virus. For maintaining the cells, maintenance medium containing EMEM with 5% foetal bovine serum (FBS, Sigma) was used.
        
The wild strain of DPV adapted in CEF upto fifth passage level was further propagated in Vero cell line and QT-35 cell line. Cell free supernatant from CEF primary culture was filtered through 0.45 μm membrane filter (Nalgene) and treated with cocktail antibiotic (Sigma, 100X) @ 10 μl/ml. A 24 hour old 60-70% confluent cell monolayer was used for infection by duck plague field strain. Growth medium from the tissue culture flask was removed; 1 ml of antibiotic treated sample was then added and the flasks were incubated at 37°C in CO₂ incubator for virus adsorption. Hank’s balanced salt solution (HBSS) was used to wash away the un-adsorbed viruses and approximately 5 ml of the maintenance medium containing 5% FBS was added. The flasks were then incubated at 37°C in a CO₂ incubator for a period of 4-5 days. The cells were monitored everyday till CPE appeared. After completion of 70-80% CPE the virus was harvested by three freeze thaw cycles. After clarification by centrifugation at 3000 rpm for 15 minutes, the virus containing fluid was aliquoted in vials and used for further passaging.
 
Confirmation of cell culture adapted DPV
 
Duck plague virus adapted in CEF, Vero as well as at QT cell line was subjected for confirmation at different passage level by PCR and s-ELISA.
 
Polymerase chain reaction (PCR)
 
Extraction of genomic DNA was done by Trizol method according to manufacturers guidelines. The 5^th, 10^th and 12^th passaged virus in various cell culture systems was tested for presence of virus by PCR assay. For detection of DNA polymerase gene UL30 specific primers Aravind et al., (2015) were used. The details of the primers used in the present study are given below.
 
 

For amplification of DNA polymerase gene a 50 µl reaction mixture containing 25 µl of master mix, forward primer (10 pmol/µl) 2 µl, reverse primer (10 pmol/µl) 2 µl, nuclease free water 13 µl and DNA template 8 µl was prepared. Thermal cycling conditions used were initial denaturation at 95°C for 3 mins followed by 30 cycles each of 94°C for 30 sec, 57°C for 1 min, 72°C for 1 min with a final extension of 72°C for 8 min. The amplified PCR products of DNA polymerase gene of DPV was confirmed by agarose gel electrophoresis in 1X Tris Acetate EDTA (TAE) buffer with 1.7% Agarose (Amresco) containing ethidium bromide (10 mg/ml). Electrophoresis was carried out at 80V for 1 hour and amplified product was visualized at 312 nm wavelength as a single compact band of expected size under UV light using a UV transilluminator (DNR, Bio-imaging system, Minilumi).

Sandwich-ELISA
 
A sandwich-ELISA (s-ELISA) was developed for the detection of virus adapted in various cell culture system (Hung et al., 2004). Purified DPV was used to raise antibody in duck as well as in rabbit using standard protocol. A 96 well microtitre plate was coated with 50 µl per well of 1:800 diluted rabbit anti-duck plague virus antibody in carbonate-bicarbonate buffer (pH9.6) and was incubated overnight at 4°C. The coated plate was washed with PBST for 3-4 times and pre-blocking was done with blocking buffer containing 3% LAH + 2% goat serum + 5% horse serum in coating buffer @ 50 µl/well. The plate was incubated at 37°C for 1 hour. The plates were washed for three times and the test samples were diluted in diluting buffer (PBST+2% Horse serum) in the ratio of 1:2 by adding 50 µl to each well. Incubation was done for 1 hour at 37°C. After thorough washing duck anti-duck plague antibody (1:300) diluted in blocking buffer was added 50 µl to all the wells and incubated for 1 hour. The plate was washed thoroughly and goat anti-duck HRPO conjugate (1:300) diluted in blocking buffer @ 50 µl was added to all the wells and incubated for 1 hour. The plates were washed and 50 µl of freshly prepared substrate (H2O2) and chromogen (OPD, Sigma was added to all the wells and allowed to react for 15 minutes. The reaction was stopped with 1M H2SO4. Reading was done using 492 nm filter. The cut-off value of each sample was calculated by subtracting the mean optical density (O.D) value of the negative control from that of the test samples. Difference of O.D value ≥ 0.1 was considered positive.

Adaptation of wild strain of DPV
 
The primary chicken embryo fibroblast (CEF) monolayer was prepared from 9-11 days old embryonated eggs. Confluent monolayer of CEF was developed within 24 hour (Fig 1). The DPV virus was passaged in CEF up to 12 passage level. CPE observed in 4^th passage comprised of vacuolation, rounding, syncytia formation and finally detachment of cells (Fig 2, 3, 4). It was seen that in 4^th and 5^th passage CPE developed within 48 hours of infection and CPE observed were vacuolation and rounding. In 6^th passage level the time of appearance of CPE was within 48 hours and CPE consisted of vacuolation, rounding, syncytia and detachment of cells. From eight passage level all characteristic CPE could be observed within 24 hours post inoculation. It was observed that the time taken for appearance of CPE decreased with passage levels. The uninfected cell monolayer retained normal cellular details throughout the passage levels. PCR was performed on 5^th, 10^th and 12^th passage level and viral nucleic acid could be detected (Fig 5). On the other hand Sandwich ELISA could detect viral antigen from 10^th passage onwards (1:16). The virus titre increased gradually with increase in passage level and in 12^th passage the titre was found to be 1:64. Comparable results were observed in a study conducted by Kumar and Ponnoose (1997) where the virus was adapted in CEF cultures by serial passaging upto 10^th passage level and reported demonstration of rounding and clumping of cells, syncytium formation, vacuolation of cytoplasm and eosinophilic intra-nuclear inclusion bodies. Similarly, Dinh et al., (2004) conducted an experiment on adaptation of DPV in CEF and observed that in 4t^h to 7^th passage level CPE was observed after 72 hours while from 8^th passage the time taken for demonstration of CFE reduced to 48 hours. Mondal et al., (2010) opined that first signs of CPE were evident at 12 hour post infection and by 48 hour more than 80% cells became rounded with vacuolation, cytoplasmic extension and detachment. Adaptation of DPV virus in CEF culture were also conducted by Doley et al., (2013) and Neher (2016) who reported that characteristic CPE appeared within 24 hours post infection and along with increase in the number of passages, the CPE appeared earlier. Complete CPE could be detected by 48 hours. Adaptation of DPV field strain in chicken embryo fibroblast cell culture at different passages was confirmed using PCR. PCR could detect the viral nucleic acid from the fourth passage itself. Similar findings were also reported by Dinh et al., (2004) where they could detect virus from CEF cell culture from 1^st passage onwards by PCR.
 

 

 

 

 

        
The details of the CPE produced, PCR confirmation and S-ELISA detection is shown in the Table 1.
 

        
A confluent monolayer of Vero cell was developed within 24 hours (Fig 6). The cell monolayer attaining 60-70% confluence was infected with CEF adapted DPV. The field strain was propagated up to 12^th passage and presence of virus was confirmed from 5^th passage onwards by PCR (Fig 5). The detection of virus in Vero cell line by S-ELISA was possible only from 10^th passage. The titre of the virus at 10^th and 12^th passage level was found to be 1:16 which was less than the titre of DPV adapted in CEF. Though the virus was detected by PCR, no prominent CPE was observed up to 12 passage level (Fig 7). The adaptation of field strain of DPV in Vero cell line at different passage level and their confirmation by PCR and ELISA is depicted in the Table 2.
 

 

 

        
The Vero cell line, being versatile in nature, could be easily maintained in the laboratory. There are many reports on adaptation of viruses of avian origin in Vero cell line like Marek’s disease (Jaikumar et al., 2001), Newcastle disease virus (Ahamed et al., 2004), Avian reo virus (Nwajei et al., 1988) and Chicken Infectious Anaemia virus (Samah et al., 2013) but there is only one  report on adaptation of DPV in Vero cell line. In the study conducted by Aravind et al., (2015), it was observed that gradual CPE could be seen from the 7^th passage onwards and from 15^th passage there was development of prominent CPE.
        
The QT-35 cells were maintained by continuous sub culturing in growth media. A confluent cell monolayer was developed within 48 hour (Fig 8). The cell monolayer attaining 60-70% was infected with CEF adapted DPV. The virus was propagated up to 12^th passage level. The characteristic CPE observed in CEF cell were not evident in QT-35 cell line, however intermittent thinning of cells followed by detachment was found from 8^th passage (Fig 9). PCR was positive from 5^th passage level and S-ELISA was done for determination of virus titre at different passages. The titre of the adapted virus at 10^th and 12^th passage was found to be 1:16. The details of the PCR confirmation, appearance of CPE and ELISA titre is shown in the Table 2.
 

 

        
There are no previous reports on adaptation of DPV in QT cell lines. However, QT cell line has been extensively used for isolation and propagation of Fowl pox virus and Adeno virus (Yadav et al., 2007; Kamal et al., 2015). The characteristic CPE observed in CEF cell were not evident in QT-35 cell line, however intermittent thinning of cells followed by detachment was found from 8^th passage onwards. The adaptation of DPV could be confirmed by performing PCR at various passage levels. PCR was positive from 5^th passage level.
        
The adaptation of duck plague virus in different cell culture systems was demonstrated by PCR. Several workers have opined that the speed, sensitivity and specificity of PCR greatly improved the diagnostic and research tool for studying of duck plague (Hansen et al., 1999; Pritchard et al., 1999. The DNA polymerase gene (UL30) was targeted because of the highly conserved nature of the gene (Xuefeng et al., 2008a; Mondal et al., 2010). The viral DNA was extracted from the cell culture fluid by trizol method and used for amplification. The amplicon size was 1510 bp (Aravind et al., 2015) and there was no nonspecific amplification or smearing.
        
In the present study a Sandwich ELISA test was standardized for the detection of virus titre in cell culture. ELISA is reported to be simple alternative to standard duck inoculation method and neutralization test for diagnosis of DPV (Chandrika et al., 1999) S-ELISA was able to detect virus at 10^th passage level in CEF, Vero and QT-35 cell line. Neher et al., (2018) have used S-ELISA for detection of DPV antigen from field samples and termed it as a rapid diagnostic test for detection of DPV antigen. Similarly S-ELISA has been used for detection of DPV antigen in field sample as well as from the CAM and allantoic fluids of inoculated birds (Tu et al., 2004, Hung et al., 2004).
        
Through this research work an attempt was made for adaptation of wild strain of DPV in both primary and continuous cell lines. The local strain was successfully adapted in various systems and the findings were found to be comparable with the observations made by other workers. It was seen that the titre of the virus adapted increased with passage level and further study is required for studying their immunogenicity and ability to be used as a vaccine candidate in future.

The authors are thankful to the Dean and Director of Research (Veterinary), Faculty of Veterinary Science, AAU, Khanapara for providing necessary facilities to carry out the research work. This study was funded by Department of Biotechnology, Government of India under DBT Twinning project on ‘‘Development of novel molecular diagnostics and improved vaccine for Duck Plague virus’’(Grant No. BT/372/NE/TBP/ 2012).