Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

The overall goal of the following experiment is to predict the co-receptor usage of HIV one for the administration of a new class of antiretroviral drugs. This is achieved by isolating the viral genomic nucleic acids, viral RNA or proviral DNA from the patient's blood following amplification of the envelope region. The viral V three region, which is the main determinant for tropism, is amplified by nested PCR.

Next, the V three amplicon is sequenced with different primers in order to generate a fasta file. Results are obtained that show the viral tropism and coreceptor antagonist susceptibility based on the false positive rate or FPR as obtained by the online interpretation tool geno to pheno coreceptor. Depending on the CORECEPTOR use, HIV viruses are classified as R five or X four.

Miri rock binds through the CCR five receptor, inhibiting the entry of R five viruses in the target cell. During the course of disease X four viruses may emerge and outgrow the R five viruses. The termination of receptor use also called tropism, is therefore mandatory prior to administration of maraviroc as demanded by EMA and FDA.

The main advantage of this technique is that one can make a parallel determination of the resistance to other antiretroviral drug classes very easily. This method allows for much more personalized medicine as the cutoffs for the tropism results may be modified according to the patient's needs. We first have the idea for this method at the beginning of the nineties when we wanted to know the molecular basis of the different cell culture growth of HIV strains and the disease progression in patients demonstrating the procedure will be data a technician from our group Prior to the start of this protocol.

Samples are collected as EDTA blood at the clinical site and sent to the laboratory to begin isolate HIV nucleic acids from 1000 microliters of whole blood serum or plasma using the mag pure compact system and nucleic acid isolation kit per manufacturer's instructions. The gained RNA or DNA is then alluded in 50 microliters TE buffer. The next step is to amplify the envelope region of the nucleic acid by reverse transcription PCR for RNA or simply PCR for DNA as outlined in the text, the reaction should generate a 1, 245 base pair long product comprising the majority of the envelope protein region to amplify the V three region of the envelope protein follow with nested PCR using five microliters from the first PCR reaction as the template.

Prepare the reactions using the master mix for nested in-house PCR as described in the text. Nested PCR running parameters are also outlined there. Following amplification of the V three region, analyze the PCR product to verify the expected product at 902 base pair in length by gel electrophoresis before sequencing.

Purify the PCR product as described in the written protocol. Once the PCR product has been purified, perform the sequencing reaction as outlined in the text upon completion of the reaction. Purify the sequencing samples using cidex G 50 superfine, and a multi-screen filter plate with durapore membrane following instructions in the written portion of this procedure to sequence the purified product, prepare the sequencer by checking the polymer and changing the buffer.

Then load the samples, set the conditions, and start the run. Following the sequencing run. The DNA STAR laser gene program can be used to align and edit the sequences.

The V three amplicon is sequenced with at least one forward and one reverse primer. Laser gene aligns the ABI files obtained from the sequencer with the reference sequences. V three, consensus B and envelope laser gene creates a consensus sequence of the analyzed sample using all the raw data available, but not the references and stores it as a FASTA file.

The FASTA file is a text file that includes a header with the name of the sample and the nucleotide sequence. To begin data interpretation and tropism prediction, upload the FASTA file into the web-based interpretation system. Geno Tono co-receptor select the FPR cutoff up to which the virus is classified as R five.

By using the German guidelines, the virus is classified as R five. When FPR is greater than or equal to 20%and as X four, when FPR is less than 12.5%select any additional clinical parameters if available, and then select, align and predict This server translates the nucleotide sequence into amino acids, aligns it with the V three consensus B sequence, and produces a subtype classification. In addition, it generates a prediction of the co-receptor usage expressed as FPR.

The output shows a graded interpretation depending on the likelihood of the co-receptor usage. For high FPR values, one is quite sure that the virus is not X four and for low FPR one is unsure of what the virus looks like if the interpretation, text and background color are green. Safe administration for Ravi Rock is indicated, whereas yellow suggests a possible low risk for using Ravi Rock, and red indicates the Ravi Rock should not be prescribed.

In addition, the server generates a PDF report that can be printed and sent to the physician. Additional data such as patient's name or date of blood extraction can be manually included using a PDF writer program. In the privacy of the user's computer specific comments can also be added.

Afterward watching this video, you should have a good understanding of how to determine the viral chops using the geno pheno receptor tool While attempting this procedure, it's important to regularly perform quality controls. Don't forget that working with HIV can be extremely hazardous, so precautions and authorized biological secure laboratories should always be used.