F Gene and N Gene based Reverse Transcription PCR for Molecular Characterization of Peste des Petits Ruminants Virus

Peste des petits ruminant (PPR) or Pseudo Rinderpest, which literally means Plague of small ruminants is an economically significant disease of sheep and goats. The disease is highly contagious in nature, and is clinically characterized by stomatitis-pneumo-enteritis complex in which morbidity reported is as high as 100 per cent and mortality reaches upto 90 per cent (Manimaran et al., 2017). Kataria et al., (2007) reported a case fatality rate of 70 per cent in sheep and 80 per cent in goats in Haryana, India.
       
Although different DNA based PCR techniques are used for routine molecular studies of microbes it is different with RNA viruses where reverse transcriptase polymerase chain reaction RT-PCR is a tool for detection of nuclear material from samples (Forsyth and Barrett 1995). Therefore, on the same lines RT-PCR is developed for precise and accurate diagnosis of PPR virus by detecting different genes likes F gene, N gene, P gene, M gene, H gene etc. Out of these F gene responsible for fusion of virus with host cell is supposed to be the important factor responsible for virulence and therefore, the primers developed for F gene by Forsyth and Barrett (1995) has been considered as most sensitive and popular tool for diagnosis of PPR. 
       
There are four distinct lineages of the PPRvirus circulating worldwide out of which lineage 4 is most common in India, excepting a solitary isolate (India/TN/92) of lineage 3 from Tamil Nadu (Shaila et al., 1989). Recent studies have revealed that most widely used F gene based PCR techniques have failed to detect PPR virus from few of the samples that had been tested positive by sandwich ELISA (Tiwari, 2004). Therefore, a need was felt to assess the suitability of other PPR virus gene targets for its detection from field samples. Considering the aforementioned points, present study was undertaken with the objectives of detection of PPR antigen in various samples by immune-capture sandwich ELISA, its confirmation by Reverse transcriptase PCR using two different gene specific primers F and N simultaneously and sequencing of PPR virus isolates to assess its lineage. 

A total of 83 samples from 31 clinically suspected animals were collected belonging to (name of districts) of Maharashtra state of India for detection of PPR virus. A total of 83 samples were collected which comprised of 20 samples from heart, liver and kidney collected in combination followed  by 19 lung samples, 16 spleen samples; 8 lymph nodes, 4 intestinal mucosa, 10 nasal swabs and 6 blood samples. The samples were collected aseptically, transported on ice in virus carrier medium and preserved in deep freeze at -20°C at Regional Disease Investigation Laboratory, Aundh, Pune, Maharashtra, till further serological and molecular processing. Nasal swabs and blood were individually processed with F gene and N gene specific PCR, and the tissue homogenates were pooled for processing with ‘Sample A’ comprising of heart, kidney, liver and intestine combined together and ‘Sample B’ comprising of combined lung, spleen and lymph node tissues for the molecular detection of PPR (Luka et al., 2011). Homogenate of tissue samples were prepared as described by Luka et al., (2011) with slight modification. Approximately 1gm each of tissue samples taken in combinations of sample ‘A’ and sample ‘B’ from each animal were individually triturated in a sterile pestle and mortar along with small quantity of glass powder. PBS containing 1000 IU/ml penicillin and 1000ug/ml streptomycin in 10 ml quantity was added and mixed with tissue (1:10). For sandwich ELISA the homogenate was prepared in 1:1 ratio, without mixing any antibiotic solution. The suspension prepared for ELISA was used directly in the reaction, whereas for PCR homogenate suspension was incubated at room temperature for 30 minutes and it was then centrifuged at 10000 RPM for 5 minutes. The supernatant was collected. The supernatant was preserved in sterile vial at -20°C until further processing. 
 
Detection of PPR by sandwich ELISA
 
Detection of PPR antigen was done by using sandwich ELISA kit developed at Rinderpest Laboratory, Division of Virology, IVRI, Mukteshwar and test was performed as per the protocol outline in the user’s manual supplied with kit.
 
Molecular Detection of PPR by RT-PCR
 
PPR virua being RNA virus the purification and extraction of viral RNA was carried out with commercial QIAamp viral mini kit (Qiagen) and quantification by spectrophotometric analysis with Purity of RNA was judged on the basis of optical density ratio at 260:280 nm. The samples with acceptable purity (i.e. ratio 1.7-2.0) were quantified using the following formula and used for reverse transcription:

 
 

                                                                                                                  
Reverse transcription for cDNA synthesis was performed as per manufacturer’s protocol (Qiagen, 2014).Synthesis of first strand DNA was performed by using Omniscript reverses transcription. Extracted template RNA solution was thawed on ice. The primer solution, 10x Buffer RT, dNTP Mix and RNase-free water was thawed at room temperature and stored on ice immediately after thawing. RNase inhibitor was diluted to a final concentration of 10 units/μl in ice-cold 1X Buffer RT and mixed carefully by vortexing for not more than 5 seconds Fresh master mix was prepared on ice and master mix was mixed thoroughly and centrifuged briefly and stored on ice. The RNA was incubated at 65°C for 5 min for denaturation and then kept immediately on ice. The template RNA was added to individual tube containing master mix and then mixed by vortexing. The tube was incubated for 60 min at 37°C. Aliquot of finished reverse transcription reaction was used in further PCR protocol for preparation of PCR Mix. Known Set of primers encoding sequences of F-gene and N-gene of PPRV were used where prior was reported to generate approximately 372 bp amplicon and the later463bp amplicon.
                           
Sequence analysis and phylogenetic studies
 
The nucleic acid sequences obtained were used for phylogenetic studies. The nucleic acid sequences were aligned with known isolates sequence available in National Centre for Biotechnology Information to know its variation, homology and phylogeny. The oligonucleotides specific to PPR F and N gene region were analysed by the BLAST sequence comparison algorithm (http://www.ncbi.nlm.nih.gov/BLAST). After individual alignment of sequence, multiple alignment of field sample was done with, sample belonging to different state, country and lineage with Clustal omega software. The aligned sequences were studied in NCBI and on the basis of per cent similarity and a phylogenetic tree was constructed by using MEGA 6 software.

A total of 31 clinically ill animals comprising of 14 sheep and 17 goats were tested during this study out of which 22 were found PPR positive by sandwich ELISA with overall 70.96 per cent positivity in 12 (85.71%) sheep and 10 (58.82%) goats with respiratory illness. These results were slightly inclined towards higher side than those being reported earlier (Chavan et al., 2009 and Mahajan et al., 2013). Our findings were in concurrence with Balamurgan et al., (2014) who had studied samples collected from different regions in India and reported higher incidence of PPR in sheep (45.66%) in comparison to goat (38.54 %). These findings slightly differed from Mahajan et al., (2013b) who had reported a higher incidence in case of goats (66.66%) than in sheep (35.71%), these differences may be because of the selection of only clinically ill animals in our case, small sample size and sheep goat population differences in geographical regions.


       
On testing for PPR viral antigen with sandwich ELISA, 60.00 per cent samples from heart, kidney and liver, 57.84 per cent from lung, 50.00 per cent spleen, 62.50 per cent of lymph nodes, 75.00 per cent of intestinal mucosa, 40.00 per cent of nasal swabs, and 66.67 per cent blood samples were found positive. Our findings were in accordance with Bhaskar et al., (2011) except for little variation in spleen and lymph nodes. The highest positivity in heart, kidney, and liver samples may be attributed to the infection followed by immunosuppression resulting in the increase of antigen load even in non-predilection sites also. The result of Balamurugan et al., (2012) was very much similar in case of goats, with highest positivity observed in Intestinal samples. 
 
Detection of PPR by F gene and N gene RT-PCR
 
In present study for the detection of PPR the samples as mentioned earlier were processed with RT-PCR by F1/F2 gene specific primers. Total 17 samples resulted positive demonstrating the ampilicon size of 372bp, however, all 6 blood samples processed resulted negative with this PCR (Plate 1). The 2 nasal swabs out of 10 revealed amplification products of 372 bp when amplified with F gene specific primers. As per the results the per cent positivity for detection of PPR total organs tested was 70.83 per cent, for nasal swabs it was 20 per cent while it was nil for the blood samples. Present findings were slightly higher when compared with Kerur (2008) who detected PPRV RNA in 50 per cent of the 48 clinical samples tested.  Our results were in concurrence with the earlier findings of scientists from India (Shaila et al., 1996; Dhar et al., 2002 and Raj et al., 2003). Of the 24 total samples tested with N1/N2 specific primers amplification products were obtained from 17 tissue samples, amplicon was not detected in blood sample while amplification product of 463bp was obtained in 2 nasal swabs processed with N gene specific primers (Plate 2). The comparable results were observed with N gene as that of F gene and the per cent positivity in tissues in detecting PPR was 70.83 per cent while for blood sample it was 0 per cent and for nasal swab it was 20 per cent. Our results were in concurrence with the results of Kerur et al., (2008) who had detected PPRV by N gene generating 12 samples positive out of 18.




 
Sequence analysis and Phylogenetic studies
 
The phylogenetic similarity between all the field isolates ranged between 91 per cent to 99 per cent for all Indian isolates presented in Gene Bank (Fig 1 and Fig 2). Whereas, when aligned with the Bangladesh 2000 and Morroco08-2 isolates the homology was found to be 91 per cent and 99 per cent respectively. Majority of the sequences obtained in this study were clustered in lineage IV.
 

 

       
The field isolates was compared against Sungri 96 commonly used as vaccine strain of lineage IV (Fig 1 and Fig 2). The homology was found out to be 98% for 2/KNP/F/Kolhapur, 5/KNP/F/Pune and 10/KNP/F/Aurangabad, it was 97% for 6/KNP/F/Ahmednagar and 91% for 7/KNP/F/Sangli. All the field isolates shared comparatively lesser homology ranging from 89 to 95 per cent with Nigeria/76/1 strain of lineage 1 and a homology ranging between 85 per cent  to  90 per cent for ICV89 (lineage 2). Our results are in concurrence with (Munir et al., 2012 and Khan 2013).
       
Upon N gene sequencing of isolates their sequences were aligned with different isolates reported from India in Gene Bank (Fig 2). Our isolates when aligned proved to be 94 per cent to 100 per cent homologous. The 100 per cent homology was seen between 10/KNP/N/Aurangabad and Revati-2006. While 7/KNP/N/Sangli showed greater homology of 97 per cent with isolate from the neighbouring countries such as China/Tibet/Geg7-30, Mym3.N.2013, and Dhaka-1/2010. Thus all the Asian isolates (lineage 4) including the isolates from the present study clustered into same group.
       
The entire sample showed a homology between 94 per cent to 99 per cent with Sungri 96 strain when sequence analysis was performed with N gene. The field isolates were also compared with lineage 1 (Nig75/1) and lineage 2 (Cote de Ivory) the identification score was between 86per cent to 88per cent in case of lineage 1, while it was 86 per cent in case of lineage 2. Our findings were concurrent with the findings of (Kerur et al., 2008 and Khan 2013).

Authors are very much thankful to Regional Disease Investigation Laboratory, Aundh, Maharashtrafor generously providing the facilities and support for completion of this work.