Reverse Transcription Process Research Articles

A Photoelectrochemical Biosensor Mediated by CRISPR/Cas13a for Direct and Specific Detection of MiRNA-21.

Direct detection of miRNA is currently limited by the complex amplification and reverse transcription processes of existing methods, leading to low sensitivity and high operational demands. Herein, we developed a CRISPR/Cas13a-mediated photoelectrochemical (PEC) biosensing platform for direct and sensitive detection of miRNA-21. The direct and specific recognition of target miRNA-21 by crRNA-21 eliminates the need for pre-amplification and reverse transcription of miRNA-21, thereby preventing signal distortion and enhancing the sensitivity and precision of target detection. When crRNA-21 binds to miRNA-21, it activates the trans-cleavage activity of CRISPR/Cas13a, leading to the non-specific cleavage of biotin-modified DNA with uracil bases (biotin-rU-DNA). This cleavage prevents the biotin-rU-DNA from being immobilized on the electrode surface. As a result, streptavidin cannot attach to the electrode via specific biotin binding, reducing spatial resistance and causing a positively correlated increase in the photocurrent response. This Cas-PEC biosensor has good analytical capabilities, linear responses between 10 fM and 10 nM, a minimum detection limit of 9 fM, and an excellent recovery rate in the analysis of real human serum samples. This work presented an innovative solution for detecting other biomarkers in bioanalysis and clinical diagnostics.

The Role of Tumor Suppressor p53 Protein in HIV-Host Cell Interactions.

The virus-host relationship is indispensable for executing successful viral infection. The pathogenesis of HIV is determined by an intricate interaction between the host and the virus for the regulation of HIV infection, thereby influencing various aspects, including the regulation of signaling pathways. High mutation rates and population heterogeneity characterize HIV with consequences for viral pathogenesis and the potential to escape the immune system and anti-viral inhibitors used in therapy. The origin of the high mutation rates exhibited by HIV may be attributed to a limited template-copied fidelity that likely operates in the cytoplasm. HIV-1 infection induces upregulation and activation of tumor suppressor p53 protein in the early stages of HIV-1 infection. p53 plays a multifaceted role in the context of HIV infection, thereby affecting viral replication. p53 is involved in maintaining genetic integrity, actively participating in various DNA repair processes through its various biochemical activities and via its ability to interact with components of the repair machinery. This report focuses on the impact of the p53 protein on the HIV-1 reverse transcription process while incorporating various incorrect and non-canonical nucleotides. The presence of functional host-coded p53 protein with proofreading-repair activities in the cytoplasm may lead to various biological outcomes.

Aptamer Screening: Current Methods and Future Trend towards Non-SELEX Approach.

Aptamers are nucleic acid sequences that specifically bind with target molecules and are vital to applications such as biosensing, drug development, disease diagnostics, etc. The traditional selection procedure of aptamers is based on the Systematic Evolution of Ligands by an Exponential Enrichment (SELEX) process, which relies on repeating cycles of screening and amplification. With the rapid development of aptamer applications, RNA and XNA aptamers draw more attention than before. But their selection is troublesome due to the necessary reverse transcription and transcription process (RNA) or low efficiency and accuracy of enzymes for amplification (XNA). In light of this, we review the recent advances in aptamer selection methods and give an outlook on future development in a non-SELEX approach, which simplifies the procedure and reduces the experimental costs. We first provide an overview of the traditional SELEX methods mostly designed for screening DNA aptamers to introduce the common tools and methods. Then a section on the current screening methods for RNA and XNA is prepared to demonstrate the efforts put into screening these aptamers and the current difficulties. We further predict that the future trend of aptamer selection lies in non-SELEX methods that do not require nucleic acid amplification. We divide non-SELEX methods into an immobilized format and non-immobilized format and discuss how high-resolution partitioning methods could facilitate the further improvement of selection efficiency and accuracy.

Genetic analyses of the structural protein E2 bovine viral diarrhea virus isolated from dairy cattle in Yogyakarta, Indonesia.

Bovine viral diarrhea (BVD), a highly pathogenic ribonucleic acid (RNA) virus, causes devastating financial losses and reproductive deaths among dairy cattle in Yogyakarta and globally. This study aimed to identify point mutations within the E2 structural protein of the acquired BVD virus (BVDV) isolates using genetic analysis. The study period shows that we performed the research in 2023. We collected 118 serum samples from 2019 to 2023, among which only 10 BVDV positive were used and 108 were negative lacking the BVDV antigen. An anti-Erns monoclonal antibody-coated protein was used in indirect antigen capture enzyme-linked immunosorbent assay (I-ACE) to detect the BVD antigen present in positive BVDV serum specimens. In the initial step of the two-step reverse transcription polymerase chain reaction, the enzyme (superscript III reverse transcriptase) and the primer (random hexamer) were used to convert the RNA of the BVDV into complementary deoxyribonucleic acid (cDNA) during the process of reverse transcription. The final step involved the amplification of the E2 gene of the resultant BVDV cDNA through gene-specific primers (E2_fwd: 5'-TGGTGGCCTTATGAGAC-3' and P7_rev: 5'-CCCATCATCACTATTTCACC-3') and enzyme (platinum taq DNA polymerase high fidelity). For conducting Sanger sequencing, those 3 BVDV-1-positive isolates (about 2.6% of all isolates) were selected as a typical specimen for each site and year between 2019 and 2023 using a proportional computation. Therefore, only two BVDV isolates with complete genomes were chosen to perform their homological and genetic analysis based on the E2 gene by means of Blast and MEGA Version 11 in addition to the Bioedit 7.2.5 program. By applying phylogenetic analysis relying on the E2 gene, a sum of 1011 nucleotides of the BVDV-1 isolates derived from each of the two BVDV-1 Indonesian isolates (n = 2) and its 23 reference BVDV strains were acquired from the National Center for Biotechnology Information (NCBI) database. The findings of the genetic analysis inside the phylogenetic tree revealed that the two BVDV Indonesian isolates were clustered into BVDV-1a subgenotype, while the reference BVDV strains were clustered into the five BVDV subgenotype, BVDV-1a (n = 6), BVDV-1b (n = 3), BVDV-1c (n = 11), BVDV-1m (n = 1), and BVDV-1n (n = 2). The branch exists in phylogenetic tree located before the division of our two BVDV isolates was divided into two branches with the same maximum bootstrap values of 99%, indicating a high degree of confidence, was seen. Next, we observed the branch near our study samples, which displayed the bootstrap value of 100, indicating that our 02 isolates were identical. In both isolates, V11 BVDV1/Indonesia/Yogyakarta/2023 and V16 BVDV1/Indonesia/Yogyakarta/2023 with GenBank accession numbers PP836388 and PP836389, respectively, conserved D7E residues were mutated as well as cysteine changed/altered into serine (S) was identified at amino acid position 201. We identified two isolates of BVDV belonging to the BVDV-1a subgenotype. Our findings indicate that the conserved D7E residues of isolates V11 BVDV1/Indonesia/Yogyakarta/2023 and V16 BVDV1/Indonesia/Yogyakarta/2023 were altered. The Indonesian BVDV isolates exhibited a cysteine to serine mutation at amino acid position 201, leads to vaccination failure, range of animal's host will increase, and diagnostic kit will not be effective.

Retroviral PBS-segment sequence and structure: Orchestrating early and late replication events

An essential regulatory hub for retroviral replication events, the 5’ untranslated region (UTR) encodes an ensemble of cis-acting replication elements that overlap in a logical manner to carry out divergent RNA activities in cells and in virions. The primer binding site (PBS) and primer activation sequence initiate the reverse transcription process in virions, yet overlap with structural elements that regulate expression of the complex viral proteome. PBS-segment also encompasses the attachment site for Integrase to cut and paste the 3’ long terminal repeat into the host chromosome to form the provirus and purine residues necessary to execute the precise stoichiometry of genome-length transcripts and spliced viral RNAs. Recent genetic mapping, cofactor affinity experiments, NMR and SAXS have elucidated that the HIV-1 PBS-segment folds into a three-way junction structure. The three-way junction structure is recognized by the host’s nuclear RNA helicase A/DHX9 (RHA). RHA tethers host trimethyl guanosine synthase 1 to the Rev/Rev responsive element (RRE)-containing RNAs for m7-guanosine Cap hyper methylation that bolsters virion infectivity significantly. The HIV-1 trimethylated (TMG) Cap licenses specialized translation of virion proteins under conditions that repress translation of the regulatory proteins. Clearly host-adaption and RNA shapeshifting comprise the fundamental basis for PBS-segment orchestrating both reverse transcription of virion RNA and the nuclear modification of m7G-Cap for biphasic translation of the complex viral proteome. These recent observations, which have exposed even greater complexity of retroviral RNA biology than previously established, are the impetus for this article. Basic research to fully comprehend the marriage of PBS-segment structures and host RNA binding proteins that carry out retroviral early and late replication events is likely to expose an immutable virus-specific therapeutic target to attenuate retrovirus proliferation.Graphical abstract

Research progress on the effect of hepatitis B virus DNA integration on antiviral therapy

Hepatitis B virus (HBV) DNA integration occurs during the reverse transcription process of HBV replication, which develops in the early stages of HBV infection and accompanies the entire disease course. The integration of HBV DNA is detrimental to the attainment of clinical cure goals and also raises the risk of developing liver cancer. Theoretically, nucleos(t)ide analogs can reduce the synthesis of new double-stranded linear DNA, but there is no clearance function for hepatocytes that have already integrated HBV. Therefore, patients with serum HBV DNA-negative conversions still have the risk of developing liver cancer. As an immunomodulatory drug, interferon can not only inhibit viral replication but also inhibit or even eliminate existing clonally amplified hepatocytes carrying integrated HBV DNA fragments. However, there are currently few studies on the effects of nucleos(t)ide analogues and interferon therapy on HBV DNA integration. Thus, large-scale clinical studies are urgently needed for further clarification.

Relative Expression of Toll-like Receptors, Cytokines and Acute Phase Protein by Real-Time PCR in Milk Somatic Cells of Subclinical Mastitis Affected Cattle

Background: Subclinical mastitis is one of the most important diseases of economic importance affecting dairy animals worldwide. The present study was planned to evaluate the level of expression of toll-like receptors, cytokines and acute phase protein in milk somatic cells during subclinical mastitis of cattle. Methods: The milk samples of subclinical mastitis-positive and healthy cattle were collected. After that, extraction of total RNA was done from milk somatic cells followed by cDNA synthesis by the process of reverse transcription. Following that qPCR was carried out and relative transcript levels were determined. Result: In the present study, a relative up-regulated expression was found in TLR-2, IL-1α, IL-10 and Hp, and down-regulation was found in TLR-4, TNF-α, IFN-α and IL-6 in the milk of cattle with subclinical mastitis as compared to healthy ones. Monitoring of cytokines entangled in the modulation of immune responses during the infection is useful in deciding cytokine markers that could be employed as a forecasting tool in the early diagnosis of subclinical mastitis.

An optimized prime editing system for efficient modification of the pig genome.

Prime editing (PE) is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-to-base conversions. However, its low efficiency hampers the application in creating novel breeds and biomedical models, especially in pigs and other important farm animals. Here, we demonstrate that the pig genome is editable using the PE system, but the editing efficiency was quite low as expected. Therefore, we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts (PEFs). First, we modified the pegRNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine, which significantly enhanced PE efficiency by improving the expression of pegRNA and targeted cleavage. Then, we targeted SAMHD1, a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that impedes the reverse transcription process in retroviruses, and found that treatment with its inhibitor, cephalosporin C zinc salt (CPC), increased PE efficiency up to 29-fold (4-fold on average), presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) in the PE system. Moreover, PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors (HDACis). Among the four HDACis tested, panobinostat was the most efficient, with an efficiency up to 122-fold (7-fold on average), partly due to the considerable HDACi-mediated increase in transgene expression. In addition, the synergistic use of the three strategies further enhanced PE efficiency in PEFs. Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.

Pre- and Post-Transcriptional Control of HBV Gene Expression: The Road Traveled towards the New Paradigm of HBx, Its Isoforms, and Their Diverse Functions.

Hepatitis B virus (HBV) is an enveloped DNA human virus belonging to the Hepadnaviridae family. Perhaps its main distinguishable characteristic is the replication of its genome through a reverse transcription process. The HBV circular genome encodes only four overlapping reading frames, encoding for the main canonical proteins named core, P, surface, and X (or HBx protein). However, pre- and post-transcriptional gene regulation diversifies the full HBV proteome into diverse isoform proteins. In line with this, hepatitis B virus X protein (HBx) is a viral multifunctional and regulatory protein of 16.5 kDa, whose canonical reading frame presents two phylogenetically conserved internal in-frame translational initiation codons, and which results as well in the expression of two divergent N-terminal smaller isoforms of 8.6 and 5.8 kDa, during translation. The canonical HBx, as well as the smaller isoform proteins, displays different roles during viral replication and subcellular localizations. In this article, we reviewed the different mechanisms of pre- and post-transcriptional regulation of protein expression that take place during viral replication. We also investigated all the past and recent evidence about HBV HBx gene regulation and its divergent N-terminal isoform proteins. Evidence has been collected for over 30 years. The accumulated evidence simply strengthens the concept of a new paradigm of the canonical HBx, and its smaller divergent N-terminal isoform proteins, not only during viral replication, but also throughout cell pathogenesis.

Advancing beyond reverse transcriptase inhibitors: The new era of hepatitis B polymerase inhibitors

Hepatitis B virus (HBV) is a genetically diverse blood-borne virus responsible for chronic hepatitis B. The HBV polymerase plays a key role in viral genome replication within the human body and has been identified as a potential drug target for chronic hepatitis B therapeutics. However, available nucleotide reverse transcriptase inhibitors only target the reverse transcriptase domain of the HBV polymerase; they also pose resistance issues and require lifelong treatment that can burden patients financially. In this study, various chemical classes are reviewed that have been developed to target different domains of the HBV polymerase: Terminal protein, which plays a vital role in the formation of the viral DNA; Reverse transcriptase, which is responsible for the synthesis of the viral DNA from RNA, and; Ribonuclease H, which is responsible for degrading the RNA strand in the RNA-DNA duplex formed during the reverse transcription process. Host factors that interact with the HBV polymerase to achieve HBV replication are also reviewed; these host factors can be targeted by inhibitors to indirectly inhibit polymerase functionality. A detailed analysis of the scope and limitations of these inhibitors from a medicinal chemistry perspective is provided. The structure-activity relationship of these inhibitors and the factors that may affect their potency and selectivity are also examined. This analysis will be useful in supporting the further development of these inhibitors and in designing new inhibitors that can inhibit HBV replication more efficiently.

HIV-1 3'-Polypurine Tract Mutations Confer Dolutegravir Resistance by Switching to an Integration-Independent Replication Mechanism via 1-LTR Circles.

Several recent studies indicate that mutations in the human immunodeficiency virus type 1 (HIV-1) 3'polypurine tract (3'PPT) motif can reduce sensitivity to the integrase inhibitor dolutegravir (DTG). Using an in vivo systematic evolution of ligands by exponential enrichment (SELEX) approach, we discovered that multiple different mutations in this viral RNA element can confer DTG resistance, suggesting that the inactivation of this critical reverse transcription element causes resistance. An analysis of the viral DNA products formed upon infection by these 3'PPT mutants revealed that they replicate without integration into the host cell genome, concomitant with an increased production of 1-LTR circles. As the replication of these virus variants is activated by the human T-lymphotropic virus 1 (HTLV-1) Tax protein, a factor that reverses epigenetic silencing of episomal HIV DNA, these data indicate that the 3'PPT-mutated viruses escape from the integrase inhibitor DTG by switching to an integration-independent replication mechanism. IMPORTANCE The integrase inhibitor DTG is a potent inhibitor of HIV replication and is currently recommended in drug regimens for people living with HIV. Whereas HIV normally escapes from antiviral drugs by the acquisition of specific mutations in the gene that encodes the targeted enzyme, mutational inactivation of the viral 3'PPT sequence, an RNA element that has a crucial role in the viral reverse transcription process, was found to allow HIV replication in the presence of DTG in cell culture experiments. While the integration of the viral DNA into the cellular genome is considered one of the hallmarks of retroviruses, including HIV, 3'PPT inactivation caused integration-independent replication, which can explain the reduced DTG sensitivity. Whether this exotic escape route can also contribute to viral escape in HIV-infected persons remains to be determined, but our results indicate that screening for 3'PPT mutations in patients that fail on DTG therapy should be considered.

Ultrasensitive and amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor powered by CRISPR/Cas13a

This study proposed a CRISPR/Cas13a-powered electrochemical multiplexed biosensor for detecting SARS-CoV-2 RNA strands. Current SARS-CoV-2 diagnostic methods, such as reverse transcription PCR (RT-PCR), are primarily based on nucleic acid amplification (NAA) and reverse transcription (RT) processes, which have been linked to significant issues such as cross-contamination and long turnaround times. Using a CRISPR/Cas13a system integrated onto an electrochemical biosensor, we present a multiplexed and NAA-free strategy for detecting SARS-CoV-2 RNA fragments. SARS-CoV-2 S and Orf1ab genes were detected in both synthetic and clinical samples. The CRISPR/Cas13a-powered biosensor achieved low detection limits of 2.5 and 4.5 ag/µL for the S and Orf1ab genes, respectively, successfully meeting the sensitivity requirement. Furthermore, the biosensor's specificity, simplicity, and universality may position it as a potential rival to RT-PCR.

Ultrafast and absolute quantification of SARS-CoV-2 on food using hydrogel RT-LAMP without pre-lysis.

With rapid growing of environmental contact infection, more and more attentions are focused on the precise and absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus on cold chain foods via point-of-care test (POCT). In this work, we propose a hydrogel-mediated reverse transcription loop-mediated isothermal amplification (RT-LAMP) for ultrafast and absolute quantification of SARS-CoV-2. Cross-linked hydrogel offers opportunities for digital single molecule amplification in nanoconfined spaces, facilitating the virus lysis, RNA reverse transcription and amplification process, which is about 3.4-fold faster than conventional bulk RT-LAMP. Ultrafast quantification of SARS-CoV-2 is accomplished in 15 min without virus pre-lysis and RNA extraction. The sensitivity can accurately quantify SARS-CoV-2 down to 0.5 copy/μL. Furthermore, the integrated system has an excellent specificity, reproducibility and storage stability, which can be also used to test SARS-CoV-2 on various cold chain fruits. The developed ultrafast and simple hydrogel RT-LAMP will be an enormous potential for surveillance of virus or other hazardous microbes in environmental, agricultural and food industry.

Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 505 million confirmed cases, including over 6 million deaths. Reference materials (RMs) of SARS-CoV-2 RNA played a crucial role in performance evaluation and quality control of testing laboratories. As the potential primary characterization method of RMs, reverse transcription digital PCR (RT-dPCR) measures the copy number of RNA, but the accuracy of reverse transcription (RT) efficiency has yet to be confirmed. This study established a method of enzymatic digestion followed by isotope dilution mass spectrometry (IDMS), which does not require an RT reaction, to quantify in vitro–transcribed SARS-CoV-2 RNA. RNA was digested to nucleotide monophosphate (NMP) within 15 min and analyzed by IDMS within 5 min. The consistency among the results of four different NMPs demonstrated the reliability of the proposed method. Compared to IDMS, the quantitative result of RT-dPCR turned out to be about 10% lower, possibly attributed to the incompleteness of the reverse transcription process. Therefore, the proposed approach could be valuable and reliable for quantifying RNA molecules and evaluating the RT efficiency of RT-based methods.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-022-04238-6.

Ultrasensitive electrochemical biosensor for microRNA-377 detection based on MXene-Au nanocomposite and G-quadruplex nano-amplification strategy

MicroRNAs (miRNAs) are considered as potential biomarkers for early diagnosis and prognostic assessment of diabetic nephropathy. In this work, an electrochemical biosensor for ultrasensitive detection of miRNA-377 was constructed based on MXene-Au nanocomposites and G-quadruplex nano-amplification strategy. As a promising nanocarrier, taking advantage of leveraging synergistic effects between Au nanoparticles (AuNPs) and MXene nanosheet, MXene-Au nanocomposites exhibited excellent electronic conductivity and provided massive active sites for DNA capture probe immobilization by Au-S bonds. AuNPs modified with Guanine-rich sequence DNA detection probes were designed as signal amplification nano-labels. Specifically, by inducing the transition of Guanine-rich detection probes to G-quadruplex, the strong affinity interaction between methylene blue and G-quadruplex could not only reflect trace concentration information of miRNA-377, but also lead to the further enhancement of electrochemical signal (2.7-fold). As a result, this newly designed biosensor exhibited superior sensing performance with a wide linear range from 10 aM to 100 pM and the limit of detection was as low as 1.35 aM. Compared with biosensors based on other nanocomposites and reported miRNA-377 biosensors, the proposed sensing platform did not require any thermal cycling or reverse transcription process, meeting the miRNA sensing requirements of convenient, sensitive, specific and stable. Furthermore, the as-constructed biosensor also displayed good selectivity, which was applied to accurate detection of miRNA-377 in human serum samples with satisfactory sensitivity, suggesting the biosensor system has promising applications in biological researches and early clinical diagnosis for diabetic nephropathy.

Test Strip Platform Spin-Off for Telomerase Activity Detection: Development of an Electrochemical Biosensor.

Telomerase overexpression has been associated directly with cancer, and the enzyme itself is recognized within the scientific community as a cancer biomarker. BIDEA’s biosensing strip (BBS) is an innovative technology capable of detecting the presence of telomerase activity (TA) using electrochemical impedance spectroscopy (EIS). This BBS is an interdigital gold (GID) electrode array similar in size and handling to a portable glucose sensor. For the detection of the biomarker, BBS was modified by the immobilization of a telomere-like single strand DNA (ssDNA) on its surface. The sensor was exposed to telomerase-positive extract from commercially available cancer cells, and the EIS spectra were measured. Telomerase recognizes the sequence of this immobilized ssDNA probe on the BBS, and the reverse transcription process that occurs in cancer cells is replicated, resulting in the ssDNA probe elongation. This surface process caused by the presence of TA generates changes in the capacitive process on the electrode array microchip surface, which is followed by EIS as the sensing tool and correlated with the presence of cancer cells. The telomerases’ total cell extraction protocol results demonstrate significant changes in the charge-transfer resistance (Rct) change rate after exposure to telomerase-positive extract with a detection limit of 2.94 × 104 cells/mL. Finally, a preliminary study with a small set of “blind” uterine biopsy samples suggests the feasibility of using the changes in the Rct magnitude change rate (Δ(ΔRct/Rcti)/Δt) to distinguish positive from negative endometrial adenocarcinoma samples by the presence or absence of TA.

Single-Particle Tracking Reveals the Interplay between HIV-1 Reverse Transcription and Uncoating.

Reverse transcription uses the reverse transcriptase enzyme to synthesize deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template. This plays an essential role in viral replication. There are still, however, many unknown facts regarding the timing and dynamic processes involved in this life stage. Here, three types of dual-fluorescence human immunodeficiency virus type-1 (HIV-1) particles were constructed with high infectivity, and the sequential process of reverse transcription was observed by real-time imaging of a single HIV-1 particle. Viral uncoating occurred at 60-120 min post infection. Subsequently, at 120-180 min post infection, the viral genome was separated into two parts and reverse-transcribed to generate a DNA product. Nevirapine (NVP), a reverse transcriptase inhibitor, can delay the dynamic process. This study revealed a delicate, sequential, and complex relationship between uncoating and reverse transcription, which may facilitate the development of antiviral drugs.

HIV-1 Reverse Transcriptase Inhibition by Major Compounds in a Kenyan Multi-Herbal Composition (CareVid™): In Vitro and In Silico Contrast.

CareVid is a multi-herbal product used in southwest Kenya as an immune booster and health tonic and has been anecdotally described as improving the condition of HIV-positive patients. The product is made up of roots, barks and whole plant of 14 African medicinal plants: Acacia nilotica (L.) Willd. ex Delile (currently, Vachelia nilotica (L.) P.J.H Hurter & Mabb.), Adenia gummifera (Harv.) Harms, Anthocleista grandiflora Gilg, Asparagus africanus Lam., Bersama abyssinica Fresen., Clematis hirsuta Guill. & Perr., Croton macrostachyus Hochst. ex Delile, Clutia robusta Pax (accepted as Clutia kilimandscharica Engl.), Dovyalis abyssinica (A. Rich.) Warb, Ekebergia capensis Sparm., Periploca linearifolia Quart.-Dill. & A. Rich., Plantago palmata Hook.f., Prunus africana Hook.f. Kalkman and Rhamnus prinoides L’Her. The objective of this study was to determine the major chemical constituents of CareVid solvent extracts and screen them for in vitro and in silico activity against the HIV-1 reverse transcriptase enzyme. To achieve this, CareVid was separately extracted using CH2Cl2, MeOH, 80% EtOH in H2O, cold H2O, hot H2O and acidified H2O (pH 1.5–3.5). The extracts were analysed using HPLC–MS equipped with UV diode array detection. HIV-1 reverse transcriptase inhibition was performed in vitro and compared to in silico HIV-1 reverse transcriptase inhibition, with the latter carried out using MOE software, placing the docking on the hydrophobic pocket in the subdomain of p66, the NNRTI pocket. The MeOH and 80% EtOH extracts showed strong in vitro HIV-1 reverse transcriptase inhibition, with an EC50 of 7 μg·mL−1. The major components were identified as sucrose, citric acid, ellagic acid, catechin 3-hexoside, epicatechin 3-hexoside, procyanidin B, hesperetin O-rutinoside, pellitorine, mangiferin, isomangiferin, 4-O-coumaroulquinic acid, ellagic acid, ellagic acid O-pentoside, crotepoxide, oleuropein, magnoflorine, tremulacin and an isomer of dammarane tetrol. Ellagic acid and procyanidin B inhibited the HIV-1 reverse transcription process at 15 and 3.2 µg/mL−1, respectively. Docking studies did not agree with in vitro results because the best scoring ligand was crotepoxide (ΔG = −8.55 kcal/mol), followed by magnoflorine (ΔG = −8.39 kcal/mol). This study showed that CareVid has contrasting in vitro and in silico activity against HIV-1 reverse transcriptase. However, the strongest in vitro inhibitors were ellagic acid and procyanidin B.

Molecular Characterization and Phylogenetic Analyses of full-length Viral Genomes From Iranian Patients With Chronic Hepatitis B Virus

Aim: Chronic hepatitis B infection is the main cause of liver complications such as hepatic failure, liver cirrhosis and hepatocellular carcinoma (HCC). In this study, we attempted to evaluate molecular characterization and phylogenetic analyses of full-length viral genomes from chronic hepatitis B virus (HBV)-infected patients. Methods: The full-length genomic sequence of the five HBV isolates from Ahvaz (city of Iran) patients was amplified, cloned in pTZ57R/T vector, sequenced and examined. Results: Phylogenetic analyses showed that all isolates belonged to genotype D (D1/D3). Serotyper tool identified ayw2 serotype in all HBV isolates. YMDE mutation was detected in an HBV isolate in the reverse transcriptase domain. Conclusion: In the present study, the analyses of full-length sequence of genome revealed that the HBV genotype D, sub-genotype D1/D3, and subtype ayw2 were predominant among Ahvaz HBV strains. As HBV genome replicates and is mediated via reverse transcription process, periodic investigations of full HBV genome are needed.

Natural Products with Inhibitory Activity against Human Immunodeficiency Virus Type 1.

Infections caused by human immunodeficiency virus (HIV) are considered one of the main public health problems worldwide. Antiretroviral therapy (ART) is the current modality of treatment for HIV-1 infection. It comprises the combined use of several drugs and can decrease the viral load and increase the CD4+ T cell count in patients with HIV-1 infection, thereby proving to be an effective modality. This therapy significantly decreases the rate of morbidity and mortality owing to acquired immunodeficiency syndrome (AIDS) and prolongs and improves the quality of life of infected patients. However, nonadherence to ART may increase viral resistance to antiretroviral drugs and transmission of drug-resistant strains of HIV. Therefore, it is necessary to continue research for compounds with anti-HIV-1 activity, exhibiting a potential for the development of an alternative or complementary therapy to ART with low cost and fewer side effects. Natural products and their derivatives represent an excellent option owing to their therapeutic potential against HIV. Currently, the derivatives of natural products available as anti-HIV-1 agents include zidovudine, an arabinonucleoside derivative of the Caribbean marine sponge (Tectitethya crypta), which inhibits the reverse transcriptase of the virus. This was the first antiviral agent approved for treatment of HIV infection. Additionally, bevirimat (isolated from Syzygium claviflorum) and calanolide A (isolated from Calophyllum sp.) are inhibitors of viral maturation and reverse transcription process, respectively. In the present review, we aimed to describe the wide repertoire of natural compounds exhibiting anti-HIV-1 activity that can be considered for designing new therapeutic strategies to curb the HIV pandemic.