Quantitation of Rabies Virus in Various Bovine Brain Structures

Quantitation of rabies virus in various bovine brain structures. Mexico is a country with considerable livestock potential. The states with the highest livestock production contain both humid tropical regions and dry regions that are at risk for rabies outbreaks due to the presence of the vampire bat, Desmodus rotundus, the main transmitter of rabies.

Detection of the rabies virus nucleoprotein N gene is primarily used for rabies diagnosis by molecular tests. Using PCR-based approaches, minimal quantities of an infectious agent can be detected in a clinical sample through the selective and repetitive amplification of a DNA nucleotide sequence. qRT-PCR is based on the detection and quantification of a molecule where fluorescence signal increases are in direct proportion to the amount of PCR product in a single reaction.

As the number of copies of the nucleic acid target increases, so does the fluorescence. Based on this, the aim of the study was to use quantitative qRT-PCR to determine the number of viral particles in different anatomical structures of bovine brains, following death, due to rabies infection. Total RNA was extracted directly from bovine brains using an organic extraction method according to the following protocol.

Use 100 milligrams of brain tissue from each anatomical structure to extract total RNA using a commercially available reagent containing guanidinium isothiocyanate. Manually homogenize a total of 100 milligrams of tissue from each structure in one milliliter of the guanidinium isothiocyanate reagent by vortexing using a Dounce homogenizer with a small pestle inside the microtube for 15 seconds at maximum speed. Incubate the samples on ice for five minutes, and then add 200 microliters of chloroform.

Mix the samples vigorously for 15 seconds. Incubate on ice for two to three minutes, and then centrifuge at 12, 000 x g for 15 minutes at four degrees centigrade. Transfer the aqueous phase to a clean tube and add 500 microliters of isopropyl alcohol.

Incubate the samples for 15 minutes at 21 degrees centigrade. Centrifuge the samples at 12, 000 x g for 10 minutes at four degrees centigrade. Aspirate the aqueaous supernatant by pipetting.

The isolated RNA will be present as a pellet in the tube. Next, wash the RNA pellet with one milliliter of 75%ethanol by pipetting and centrifuge at 7, 500 x g for five minutes at four degrees centigrade. Decant the supernatant and air dry the RNA pellet at room temperature, 21 degrees centigrade.

Next, dilute the pellet in 50 microliters of nuclease-free water. Determine the RNA concentration and quality at 260 nanometers using a spectrophotometer. In vitro transcription generates mRNA of a target gene.

Use a primer pair that amplifies the complete RABV N gene, and one that is used as a positive control for the qRT-PCR assay. These primers are designed to amplify the complete RABV N gene, and to add a promoter that recognizes the T7 polymerase. To amplify the whole N gene RABV, use the primers trans and RAB forward and reverse, and a high fidelity PCR kit.

For each reaction, prepare a PCR master mix by adding to a clean PCR tube reaction buffer with 10 micromolar of each primer and 0.5 units of high fidelity polymerase. To use the PCR product as a template for the in vitro synthesis and to remove components of the enzymatic reaction, purify the PCR product using a column-based concentrator kit, according to the manufacturer's instructions. And then quantify using a spectrophotometer.

For RNA synthesis, use an in vitro transcription kit with the following reaction mixture:five microliters T7 transcription 5X buffer, 1.9 microliters of each triphosphate nucleotide, 10 micrograms of purified RABV N DNA, and 2.5 microliters of the enzyme mixture. Next, incubate the mixture at 37 degrees centigrade for four hours. Then, add one microliter of one unit per microgram Rnase-Free DNase to the reaction mixture and incubate for 15 minutes at 37 degrees centigrade to eliminate DNA contamination.

Purify the in vitro transcribed RNA and then concentrate it using phenol:chloroform:isoamyl alcohol at a ratio of 25:24:1. Resuspend the purified RNA pellet into 70 microliters of TE buffer, and then store it at 80 degrees centigrade until use. Repeat the PCR protocol until approximately five micrograms of PCR product is obtained.

After purification and concentration, the in vitro transcribed N gene mRNA will be present at a concentration of 4.711 micrograms per microliter. Confirm that the in vitro transcribed mRNA corresponds to the N gene following step five of this protocol, and use this as a positive control for the real-time reverse transcription polymerase chain reaction, qRT-PCR. For real-time reverse transcription polymerase chain reaction, use an in vitro mRNA transcript in coding the complete N gene as a positive control.

Along with a commercial one-step qRT-PCR kit, using hybridization probes according to the manufacturer's instructions. Use the probe in primers described by Coronado and co-worker 2010 to detect a conserved region of the RABV using the following thermal cycling conditions. Perform serial dilutions of the in vitro transcribed mRNA by serially pipetting one microgram per microliter of mRNA into tubes until six decoupled dilutions are obtained.

Prepare tubes at a 100 microliter volume in triplicate for use in creating the standard curve. Perform qRT-PCR as described previously. Perform qRT-PCR in a thermal cycler with a rotor by processing the various brain samples simultaneously with the running reactions for creating a standard curve and using positive controls, negative controls, and supernatants isolated from cell culture.

To evaluate the limit of detection, test efficacy, and sensitivity of the test, use a standard curve in triplicate under the same conditions. For the detection of RABV gene N, use the RNA from field samples, positive controls, negative controls, and cell culture supernatants to perform qRT-PCR using the PCR kit described previously. To determine the number of copies of the RABV genome present in the bovine brain samples, obtain CT data from the standard curve and biological samples, and from those interpolated from the calibration curve equation This figure shows bovine brain tissues exhibiting positive staining according to the direct immunofluorescence.

The results show 100%100%83.3%66%and 50%positivity for RABV in the cortex, thalamus, medulla, pons, and horn respectively. These results confirm the previous results, and at least three of the structures dissected from each brain were positive for RABV. On the other hand, the equation of the standard curve shows the relationship between the amount of RABV genetic content in the sample and the size of the PCR amplified fragment.

The amount of RABV genetic content in nanograms was deduced by interpolation of the CT values in the calibration curve using the equation presented in this figure. Using this equation, the detection limit of one times 10 to the fifth micrograms per microliter of viral RNA was found to correspond to 36 copies of the RABV genome. These results demonstrate that the assay is sensitive and can be used to detect the virus in samples that contain a low number of copies of RABV N gene.

The efficiency of the assay was calculated using the slope of the standard curve, which was 3.28. The samples used for qRT-PCR showed amplification of the RABV N gene. To determine the number of viral copies present, the CT values for each sample were interpolated using the calibration curve equation.

The results obtained in nanograms was substituted into the formula described here. This table shows the comparative results between qRT-PCR and DFA. The results of qRT-PCR assays were consistent with those obtained using DFA.

According to the results obtained in the qRT-PCR test in cases C and D, the thalamus is the structure that possesses the highest number of copies of the RABV N gene. This suggests that early infection of hypothalamic and thalamic neurons is important in the development of the disease, given that these neurons control the vegetative functions of an animal. The copy numbers of the RABV N gene that were detected in each structure of each sample are:the sensitivity and specificity of the qRT-PCR test we're both 100%as all samples were positive.

This result is consistent with the initial diagnoses of the samples. qRT-PCR is a highly sensitive technique. Some of the steps are critical to obtain the best results.

One of these is the proper handling of samples for RNA extraction. This step is crucial as RNA is easily degraded and the results could therefore be affected. Consider maintaining the sample in cold conditions, approximately four degrees centigrade during the process.

In addition, consider that several rounds of PCR application are carried out as mentioned in in vitro transcription. The qRT-PCR assay conducted in this study could detect as few as 36.3 copies of the RABV N gene per milligram of tissue. The most suitable brain structures for qRT-PCR included the cortex and thalamus.

This is based on the observations that higher concentrations of the RABV N gene were detected in these structures, and that they were also found to be positive using the RABV reference test. The test was able to detect very low concentrations, 0.00023 times 10 to the ninth copies of the virus in various samples. In addition to quantifying the viral particles in the sample, the test appears to provide a good alternative diagnostic and research tool for the detection of RABV presented here.