Detection of Segmented Viruses using RNA-PAGE and RT-PCR
Basanti Brar1, Sumnil Marwaha2; Prasad Minakshi3*
1HABITAT, GIPPCL, Biofertilizer Production and Technology Centre, CCS Haryana Agricultural University, Hisar, Haryana, India; 2ICAR- National Research Centre on Camel, Bikaner; 3*Department of Animal Biotechnology, LUVAS, Hisar, India
Corresponding authors email: minakshi.abt@gmail.com
Abstract
Majority of the viruses are consisted of monopartite genomes, whereas segmentation is also a much more common phenomenon seen in RNA viruses. In a group of viruses (all RNA viruses), the linear genome splits into two or more segments. The RNA viruses with genetic material have segmented genomes are influenza viruses, birnaviruses, picobirnaviruses, arenaviruses, bunyaviruses, bluetongue virus and rotaviruses. Here, we mainly focus on BTV, rotavirus and picobirnavirus. Bluetongue virus (BTV) is an arthropod-borne virus (arbovirus) transmitted between its ruminant hosts via the bites of haematophagous midges of the genus Culicoides spp. Bluetongue (BT) was first reported more than 125 years ago at a time European breeds of sheep were introduced into southern Africa. BT viruses have been identified in many tropical and temperate areas of the world having 10 segments in the genome. Bluetongue is a non-contagious disease of domestic and wild ruminants caused by a virus within the Orbivirus genus of the family Reoviridae and transmitted by Culicoides biting midges. Rotavirus belongs to the family Reoviridae under the genus Rotavirus having segmented genome, which can be separated in to discrete 11 segments by RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The picobirna virus (PBV) is a small, non-enveloped (33-35 nm) bisegmented novel double stranded RNA (dsRNA) virus belongs to order diplornavirales under family picobirnaviridae. Here we discuss the detection BTV, rotavirus and picobirnavirus by RNA PAGE and RT-PCR. During electrophoresis through the gel, these negatively charged macromolecules separate according to their size. The patterns of dsRNA can be visualized in the gel after staining with silver nitrate.
RNA-PAGE and RT-PCR of cell culture grown BTV isolates
RNA-PAGE has been evaluated as a screening test for the detection of bluetongue viruses and viral genomic diversity. Migration pattern of BTV genomic RNA revealed the classical 10 segments with 3:3:3:1 migration pattern in the above mentioned isolate of BTV TN-09CIBT indicating that the sample was Positive for BTV (Fig. 1-a).
Viral genomic RNA was extracted by trizol method and confirmed as BTV in RNA-PAGE. This RNA was used as a template for cDNA synthesis by the process of reverse transcription using random decamer primers and reverse transcriptase enzyme. The cDNA was subsequently used for amplification of partial ns1 gene PCR.
Bluetongue NS1 specific primers targeting ns1 gene showed an expected intense band of 274 bp without any non-specific amplification when observed in 1.5 % agarose gel (Fig. 1-b). Also the NS1 full length PCR purified product was diluted tenfold and then subjected to PCR to detect the detection limit of PCR (Fig. 2) which corresponds to 144 x 10-5 ng. In terms of copy numbers, it comes to 8.04 x 105copies.
Fig. 2-a): Electrophoretic migration pattern (3:3:3:1) of genomic dsRNA segments of BT TN01/CIBT/Hisar in PAGE gel; 2-b) BTV specific PCR, used to target non-structural (ns1) gene of BTV. Lane 1: DNA ladder, 2: Positive control, 3: Negative control, 4: TN-09-CIBT
Fig. 2- PCR of NS1 gene, tenfold dilutions of cDNA to know the detection limit of RT-PCR. Lane1: 100bp ladder, Lane 2: Negative template control, Lane9: Positive control, Lane3-8: serial tenfold dilution.
RNA-PAGE and RT-PCR of rotavirus and picobirnavirus
The segmented dsRNA genome of the virus was analyzed by RNA-PAGE. Eight percent gel was prepared by Acrylamide and bis-acrylamide. The samples were loaded and electrophoresis was carried out at a constant voltage of 100 V/cm in 1X Tris- glycine buffer till the dye came out of the gel. The gel was stained by silver nitrate for 30 min on gentle shaking platform. RNA bands were visualized in developer by manual shaking (fig 3). The reaction was stopped by the stop solution in which gel was kept for 15 min and then stored in 10 % ethanol.
- Rota virus (10 segments) (B) Multiple infection (C) Picobirna (2 segments)
Fig. 3: Migration pattern of genomic RNA segments of different multiple infected suspected isolates from bovine in RNA-PAGE.
cDNA synthesis was carried out in a 20 µl reaction mixture for both picobirnavirus and rotavirus. RNA and primer mixture was heated at 99ºC for 5 min in thermalcycler (Bioradi-cycler and eppendorf master cycler gradientTM, Germany), snap chilled on ice and followed by addition of Mo-MuLV-RT 1.0 µl (200 units / µl), 5X RT buffer (5.0 µl), 100 mM dNTPs (0.5 µl) and NFW (3.5 µl) in reaction tube. After allowing the primers to anneal at 25ºC for 10 min, reverse transcription was carried out at 37ºC for 60 min in thermal cycler. The reverse transcriptase was heat inactivated at 70ºC for 10 minutes. The cDNA was stored at -20ºC till further use.
The cDNA of each positive Picobirna and rotavirus isolate was amplified using their specific primer pair. PCR reaction (20 µl) was carried out with Qiagen Top Taq (10 µl), Forward and Reverse (0.5 µl) each primer, Nuclease Free Water (7 µl) and cDNA (2 µl)
The reagents were mixed, spun and PCR was carried out in thermal-cycler using different standardized incubation conditions. The numbers of PCR cycle were 30 for all primers. Amplified DNA fragments of Picobirna GpI with PicoB25F and PicoB43R primers resulted in the expected product of 201 bp (Fig. 4) and VP 4 gene of rotavirus resulted in to expected 855 bp amplicon (fig. 5).
Fig. 4: Amplification of bovine Picobirna GpI viral RNA by RT-PCR with primer PicoB25F and PicoB43R (Malik et al 2014). Lanes: 1: 100bp Ladder; 2: Positive control for Picobirna; 3-19: Positive Field Sample for Picobirna genotypeI; 20: NTC. The figure on left side 201 bp indicates size of the amplicons.
Figure 5: Amplification of Rota VP4 gene with primer VP4F and VP4R design by Falcone et al 1999. Lanes: 1-100 bp DNA ladder; 5,6,7,8,9,10,16,17,19,22,25: Rota positive fecal samples; 1,2,3,4,11,12,13,14,15,18,20,21,23,24: Negative samples. The figure on right side 855 bp indicates size of the amplicons.
Conclusion
RNA- PAGE and RT-PCR play very important role in the detection of segmented viruses.
