Quantitation of Rabies Virus in Various Bovine Brain Structures

Summary

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Abstract

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon’s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

Introduction

Rabies can be confirmed ante-mortem and post-mortem by various techniques that enable the detection of viral nucleic acids in the brain, skin, urine, or saliva¹. Detection of the rabies virus nucleoprotein (N) gene is primarily used for rabies diagnosis by molecular tests. This gene is also used for viral genotyping. Rabies can be diagnosed in animals using any portion of the affected brain; however, to exclude the possibility of rabies, tissue from at least two regions in the brain must be tested². Several diagnostic methods exist for rabies detection in animals; however, the direct immunofluo....

Protocol

This study was approved by and conducted in strict accordance with the recommendations for the use of animals provided by the Institutional Animal Care and Use Committee (IACUC) of the Centro Nacional de Investigación Disciplinaria en Microbiología Animal (CENID-MA).

1. Samples

1. Use different areas of the brain dissected from 6 different rabies-positive bovine brains for RT-PCR analysis.

2. Direct fluorescent antibodies (DFAs) to confirm rabies

NOTE: Detection of the rabies antigen by DFA is a qualitative method to determine the presence of the rabies nucleopr....

Results

DFA results showed 100%, 100%, 83.3%, 66%, and 50% positivity for RABV in the cortex, thalamus, medulla, pons, and horn, respectively. These results confirmed the previous results, and at least three of the structures dissected from each brain were positive for RABV. A representative positive DAF staining is shown in Figure 1.

Figure 2 shows the amplification of a fragment of the RABV N gene (step 5.5) with the primers first ones repo.......

Discussion

Previous studies have shown that DFA can only detect RABV within seven days of the sample being stored at room temperature (21 °C)¹⁴. In contrast, this work demonstrated that the sensitivity of RT-PCR begins to decrease after the samples have been exposed to room temperature for 12 days. Therefore, the RABV genome can be detected by qRT-PCR in samples exposed to room temperature for up to 23 days. This demonstrates that the sensitivity of qRT-PCR is relatively higher for more highly decompose.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chloroform	SIGMA	C7559	Facilitates recovery of the aqueous phase of PCRs which have been overlaid with mineral oil.
DNA Clean & Concentrator-500	Zimo	D4031	The DNA Clean & Concentrator-500 (DCC-500) is designed for the rapid, large format purification and concentration of up to 500 µg of high quality DNA from samples including large-scale restriction endonuclease digestions and impure DNA preparations.
Ethanol	Amresco	193-500	Purification of nucleic acids
FastStart High Fidelity PCR System kit, dNTPack	Roche	3553400001	High fidelity enzyme for the amplification of PCR products avoiding random base changes
GelDoc XR	BioRad	XR+	Analyzes larger protein and DNA gels
GelRed	Biotium	41003	A new generation of nucleic acid gel stains, they possess novel chemical features designed to minimize the chance for the dyes to interact with nucleic acids in living cells.
iCycler Thermal Cycler Gradient	BioRad	582BR	Temperature can be monitored and controlled by instrument algorithm, in-sample probe, or sample block modes
iTaq Universal Probes One-Step Kit	BioRad	1725141	Reaction mixture to carry out PCR reactions in real time using TaqMan type hybridization probes
Isopropyl alcohol	Amresco	0918-500	Precipitation of nucleic acids
QIAquick gel extraction kit	Qiagen	28706	QIAquick Kits contain a silica membrane assembly for binding of DNA in high-salt buffer and elution with low-salt buffer or water. The purification procedure removes primers, nucleotides, enzymes, mineral oil, salts, agarose, ethidium bromide, and other impurities from DNA samples
M-MLV Reverse Transcriptase	Invitrogen	28025-021	Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) uses singlestranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand.
NanoDrop 2000	Thermo-Scientific	ND2000	Microvolume Spectrophotometer
Oligo(dT)18 primer	Invitrogen	SO132	The oligo (dT)18 primer is a synthetic single-stranded 18-mer oligonucleotide with 5'- and 3'-hydroxyl ends.
RiboMAX Large Scale RNA Production Systems kit SP6 and T7	Promega	P1300	In vitro transcription reactions are used to synthesize microgram amounts of RNA probes from recombinant DNA templates. Most transcription reactions designed to generate RNA probes are optimized to maximize incorporation of radiolabeled ribonucleotides rather than to produce large amounts of RNA
Taq DNA Polymerase	Invitrogen	#EP0402	Taq DNA Polymerase is a highly thermostable DNA polymerase of the thermophilic bacterium Thermus aquaticus. The enzyme catalyzes 5’ and 3’ synthesis of DNA
TRIzol reagent	Invitrogen	15596026	The TRIzol reagent is a complete, ready-to-use reagent, designed for the isolation of high quality total RNA or the simultaneous isolation of RNA, DNA and proteins from a variety of biological samples.