Reverse Transcriptase Assay Research Articles

Monitoring of urothelial cancer disease status after treatment by digital droplet PCR liquid biopsy assays

ObjectivesReal-time monitoring of disease status would be beneficial for timely decision making in the treatment of urothelial cancer (UC), and may accelerate the evaluation of clinical trials. Use of cell free tumor DNA (cftDNA) as a biomarker in liquid biopsy is minimally invasive and its successful use has been reported in various cancer types, including UC. The objective of this study was to evaluate the use of digital droplet PCR (ddPCR)-based assays to monitor UC after treatment. Method and materialsBlood, urine and matching formalin fixed, paraffin embedded diagnostic specimens were collected from 20 patients diagnosed with stage T1 (n = 2) and T2/T3 (n = 18) disease. SNaPshot assays, Sanger sequencing and whole exome sequencing were used to identify tumor-specific mutations, and somatic mutation status was confirmed using patient-matched DNAs extracted from buffy coats and peripheral blood mononucleocytes. The ddPCR assays of the tumor-specific mutations were used to detect the fractional abundance of cftDNA in plasma and urine. ResultsSNaPshot and Sanger sequencing identified point mutations in 70% of the patients that were assayable by ddPCR. Cases of remission and relapse monitored by assays for PIK3CA E542K and TP53 Y163C mutations in plasma and urine concurred with clinical observations up to 48 months from the start of chemotherapy. A new ddPCR assay for the telomerase reverse transcriptase (TERT) promoter (-124) mutation was developed. The TERT assay was able to detect mutations in cases below the limit of detection by SNaPshot. Whole exome sequencing identified a novel mutation, CNTNAP4 G727*. A ddPCR assay designed to detect this mutation was able to distinguish mutant from wild-type alleles. ConclusionsThe study demonstrated that ddPCR assays could be used to detect cftDNA in liquid biopsy monitoring of the post-therapy disease status in patients with UC. Overall, 70% of the patients in our study harbored mutations that were assayable by ddPCR.

Synthesis, molecular docking simulation, and biological evaluation studies of novel amide and ether conjugates of 2,3‐diaryl‐1,3‐thiazolidin‐4‐ones

AbstractWith an aim to develop potent lead molecules as a novel class of reverse transcriptase (RT) inhibitors, we have synthesized amide and ether conjugates of 2,3‐diaryl‐1,3‐thiazolidin‐4‐one derivatives. The compounds 9a and 9f exhibited IC50 values of 0.21113 ± 0.013μM and 12.6804 ± 0.062μM respectively from the in vitro human immunodeficiency virus type 1 (HIV‐1) RT assay. None of the compounds showed toxicity towards peripheral blood mononuclear cells (PBMC). Structure‐activity relationship (SAR) studies were performed for the synthesized compounds in order to estimate the effect of the substitution pattern on the RT inhibition potency. In silico docking studies revealed that two or more interactions are necessary for significant activity.

Anti-HIV-1 activities of constituents from the rhizomes of Boesenbergia thorelii

A new lignan, thoreliin A (1), and a new bisnorlignan, thoreliin B (2), were isolated from a MeOH extract of the rhizomes of Boesenbergia thorelii. In addition, the known bisnorlignans 3 and 4, neolignan 5, phenylpropanoids 6–15, as well as benzenoids 18–21 were also obtained from the same source. The structures were elucidated based on their spectroscopic data. By single crystal X-ray analysis, the relative stereochemistry of 1 was confirmed. All isolated compounds were evaluated for anti-HIV-1 activities. Among them, thoreliin A (1) exhibited anti-HIV-1 activities on both HIV-1 reverse transcriptase (41.43% inhibition at 200 μg/mL) and syncytium reduction assays (EC50 20.6 μM, SI 3.7), while compounds 3–6, 9 and 11–21 showed anti-HIV-1 activity only in the anti-syncytium assay (EC50 6.6–454.1 μM, SI >1.32–7.75).

The duck EB66® cell substrate reveals a novel retrotransposon

During the establishment of the duck EB66® cell line as a new cell substrate for vaccine production in industry, a very low level of reverse transcriptase (RT) activity was detected in the culture supernatant by product-enhanced RT assay but a whole battery of tests failed to evidence infectious particles. Results from extensive biochemical and physical testing demonstrated that RT activity was associated to an intracellular, non-enveloped and dense structure different from an infectious retroviruses.In silico analysis of Anas platyrhynchos genome revealed that the most likely candidates for encoding a ribonucleoprotein (RNP)-associated RT were nine copies of chicken repeat 1 (CR1)-like elements, belonging to the non-long terminal repeat retrotransposons. The presence of the full length Anas platyrhynchos chicken repeat 1-like sequence (APCR1) was confirmed in EB66® cells and the related ribonucleic acid was present in the RT-containing fraction of EB66® cells.

The Spirocyclic Imine from a Marine Benthic Dinoflagellate, Portimine, Is a Potent Anti-Human Immunodeficiency Virus Type 1 Therapeutic Lead Compound.

In this study, we aimed to find chemicals from lower sea animals with defensive effects against human immunodeficiency virus type 1 (HIV-1). A library of marine natural products consisting of 80 compounds was screened for activity against HIV-1 infection using a luciferase-encoding HIV-1 vector. We identified five compounds that decreased luciferase activity in the vector-inoculated cells. In particular, portimine, isolated from the benthic dinoflagellate Vulcanodinium rugosum, exhibited significant anti-HIV-1 activity. Portimine inhibited viral infection with an 50% inhibitory concentration (IC50) value of 4.1 nM and had no cytotoxic effect on the host cells at concentrations less than 200 nM. Portimine also inhibited vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped HIV-1 vector infection. This result suggested that portimine mainly targeted HIV-1 Gag or Pol protein. To analyse which replication steps portimine affects, luciferase sequences were amplified by semi-quantitative PCR in total DNA. This analysis revealed that portimine inhibits HIV-1 vector infection before or at the reverse transcription step. Portimine has also been shown to have a direct effect on reverse transcriptase using an in vitro reverse transcriptase assay. Portimine efficiently inhibited HIV-1 replication and is a potent lead compound for developing novel therapeutic drugs against HIV-1-induced diseases.

A New Ellagic Acid From the Leaves and Twigs of Irvingia malayana

A phytochemical investigation of the leaves and twigs of Irvingia malayana led to the isolation of a new 3,3′,4′-tri- O-methylellagic acid-6″-acetoxy-4- O-β-glucoside (1), along with 3,3′,4′-tri- O-methylellagic acid (2), 3,3′-di- O-methylellagic acid-4- O-β-xyloside (3), 3,3′,4′-tri- O-methylellagic acid-4-β -O-glucoside (4), friedelin (5), friedelinol (6), methyl-3,4,5-trihydroxybenzoate (7), 5,7,4′-trihydroxyflavone-8- C-β-glucopyranoside (8), 5,7,3′,4′-tetrahydroxyflavone-8- C-β-glucopyranoside (9), and 5,3′,4′-trihydroxyflavone-6- C-β-glucopyranoside (10). Their structures were elucidated by means of spectroscopic techniques and direct comparison with literature data. Compounds 4 and 7 showed weak cytotoxic activity against a panel of mammalian cancerous cell lines. Furthermore, compounds 1, 2, 4, and 9 exhibited significant inhibitory activity in the syncytium inhibition assay, whereas compounds 8 and 9 displayed moderate activity in the HIV 1 reverse transcriptase assay.

Inhibition of LINE-1 Retrotransposition by Capsaicin.

Long interspersed nuclear element 1 (LINE-1 or L1) is a non-long terminal repeat (LTR) retrotransposon that constitutes approximately 17% of the human genome. Since approximately 100 copies are still competent for retrotransposition to other genomic loci, dysregulated retrotransposition of L1 is considered to be a major risk factor of endogenous mutagenesis in humans. Thus, it is important to find drugs to regulate this process. Although various chemicals are reportedly capable of affecting L1 retrotransposition, it is poorly understood whether phytochemicals modulate L1 retrotransposition. Here, we screened a library of compounds that were derived from phytochemicals for reverse transcriptase (RT) inhibition with an in vitro RT assay. We identified capsaicin as a novel RT inhibitor that also suppressed L1 retrotransposition. The inhibitory effect of capsaicin on L1 retrotransposition was mediated neither through its receptor, nor through its modulation of the L1 promoter and/or antisense promoter activity, excluding the possibility that capsaicin indirectly affected L1 retrotransposition. Collectively, capsaicin suppressed L1 retrotransposition most likely by inhibiting the RT activity of L1 ORF2p, which is the L1-encoded RT responsible for L1 retrotransposition. Given that L1-mediated mutagenesis can cause tumorigenesis, our findings suggest the potential of capsaicin for suppressing cancer development.

Novel splicing in IGFN1 intron 15 and role of stable G-quadruplex in the regulation of splicing in renal cell carcinoma

The IGFN1 (Immunoglobulin-Like And Fibronectin Type III Domain Containing 1) gene has a role in skeletal muscle function and is also involved in metastatic breast cancer, and the isoforms with three N-terminal globular domains are sufficient for its function in skeletal muscle. Two novel splicing isoforms of IGFN1 have been identified in renal cell carcinoma (RCC), one with 5’exon extension and an isoform with a novel exon. The role of G-quadruplex, a non-B DNA, was explored for the splicing alteration of IGFN1 in RCC. G-quadruplexes are the secondary structures acquired by stacking of G-quartets by Hoogsteen hydrogen bonding in DNA and RNA. IGFN1 has intronic potential G-quadruplex forming sequence (PQS) folding into G-quadruplex and is studied for its involvement in aberrant splicing. A PQS in the intron 15 of IGFN1 gene was observed in our in silico analysis by QGRS mapper and non BdB web servers. We observed PQS folds into stable G-quadruplex structure in gel shift assay and circular dichroism (CD) spectroscopy in the presence of G-quadruplex stabilizing agents Pyridostatin (PDS) and KCl, respectively. G-quadruplex formation site with single base resolution was mapped by Sanger sequencing of the plasmid constructs harbouring the cloned PQS and its mutant. This stable G-quadruplex inhibits reverse transcriptase and taq polymerase in reverse transcriptase & PCR stop assays. PDS changes the different splicing isoforms of IGFN1 in UOK146 cell line, displaying involvement of intronic G-quadruplex in IGFN1 splicing. These results lead us to propose that a stable G-quadruplex structure is formed in IGFN1 intron and a reason behind IGFN1 aberrant splicing which could be targeted for therapeutic intervention.

Anti-HIV and cytotoxic biphenyls, benzophenones and xanthones from stems, leaves and twigs of Garcinia speciosa

Eleven previously undescribed compounds, including four benzophenones (garciosones A−D), four xanthones (garciosones E−H) and three biphenyls (garciosines A−C), along with eighteen known compounds were isolated from the stems, leaves and twigs of Garcinia speciosa Wall. (Clusiaceae). Their structures were established by extensive spectroscopic analysis. For garciosines A−C, the structures were confirmed by single crystal X-ray diffraction analysis. Most of the isolated compounds were evaluated for their cytotoxic activity and anti-HIV-1 activity using the syncytium inhibition assay and HIV-1 reverse transcriptase (RT) assay. The known compounds, 4,6,3′,4′-tetrahydroxy-2-methoxybenzophenone and macluraxanthone, displayed significant cytotoxic activity with the ED50 in the range of 1.85–11.76 μM. 1,5-Dihydroxyxanthone exhibited the most potent anti-HIV activity against syncytium formation with EC50 < 17.13 μM (SI > 25.28) and 2-(3,3-dimethylallyl)-1,3,7-trihydroxyxanthone was the most active compound in the HIV-1 reverse transcriptase assay with IC50 value of 58.24 μM. Structure-activity relationship of some isolated compounds were also discussed.

Polycyclic polyprenylated acylphloroglucinols and biphenyl derivatives from the roots of Garcinia nuntasaenii Ngerns. & Suddee

Seven previously undescribed compounds, including three polycyclic polyprenylated acylphloroglucinols (garcinuntins A–C), three biphenyl derivatives (garcinuntabiphenyls A–C) and a lanostane triterpene (garcinuntine), along with thirteen known compounds were isolated from the root of Garcinia nuntasaenii Ngerns. & Suddee. Their structures were elucidated on the basis of spectroscopic techniques. For garcinuntins A–C, the absolute configurations were confirmed by the combination of single X-ray crystallography and ECD calculations. Anti-HIV activity using anti-HIV-1 reverse transcriptase and syncytium inhibition assays, and cytotoxic activity against a panel of cultured mammalian cancer cell lines of isolated compounds were investigated.

Cytotoxic, anti-HIV–1 and anti-inflammatory activities of lanostanes from fruits of Garcinia speciosa

Bioassays-guided isolation of the hexanes and ethyl acetate extracts of the fruits of Garcinia speciosa led to the isolation of three new lanostane triterpenes 1–3 together with three related known derivatives 4–6. The new structures elucidated on the basis of spectroscopic and X-ray diffraction techniques were identified to be methyl (22E,24E)-9α-hydroxy-3-oxolanosta-22,24-dien-26-oate (1), (22E,24E)-9α,11β-dihydroxy-3-oxolanosta-22,24-dien-26-oic acid (2), and (24E)-23-acetoxy-3-oxolanosta-9,24-dien-26-oic acid (3), namely garciosaterpenes D, E and F, respectively. All compounds were evaluated for their cytotoxic and anti-inflammatory activities along with anti-HIV–1 activity. Most of the compounds were found inactive in the cytotoxic assay except for compound 6 which exhibited selective cytotoxicity against a KB cell line. All compounds showed significant inhibitory activities against HIV–1 reverse transcriptase (RT) assay and in the syncytium inhibition assay. Compounds 1, 5 and 6 displayed promising inhibitory activity in anti-inflammatory process.

Tolerance limits and tolerance intervals for ratios of normal random variables using a bootstrap calibration

This paper addresses the problem of deriving one-sided tolerance limits and two-sided tolerance intervals for a ratio of two random variables that follow a bivariate normal distribution, or a lognormal/normal distribution. The methodology that is developed uses nonparametric tolerance limits based on a parametric bootstrap sample, coupled with a bootstrap calibration in order to improve accuracy. The methodology is also adopted for computing confidence limits for the median of the ratio random variable. Numerical results are reported to demonstrate the accuracy of the proposed approach. The methodology is illustrated using examples where ratio random variables are of interest: an example on the radioactivity count in reverse transcriptase assays and an example from the area of cost-effectiveness analysis in health economics.

Intranasal Administration of Integrase Defective Lentiviral Vectors Expressing mAbs Protects from H5 Influenza Virus Challenge In Vivo

BackgroundDespite medical advances, we are often unable to rapidly protect non-immune populations from infectious agents. Passive immunotherapy is a fast method of protection, but large-scale administration of monoclonal antibodies (mAbs) in unpractical. The delivery of mAbs using a viral vector can be an attractive alternative to direct mAbs injection. Integrase-defective lentiviral vectors (IDLV) have several advantages including the absence of pre-existing anti-vector immunity and the safety features of non-integration and non-replication. IDLV are maintained in non-dividing cells, and can express steady levels of functional proteins in vivo. We engineered IDLV to express mAbs against the influenza A virus (IAV) hemagglutinin, and tested their ability to protect from IAV in vivo.MethodsIDLV were produced by co-transfection of transfer, packaging, and envelope plasmids in 293T cells and purification on sucrose gradients. IDLV were normalized using a colorimetric reverse transcriptase assay. Plasmid expressing mAb VN04-2 was provided by B. Hanson. mAb in the supernatant of transduced cells were detected by western blot and quantified by the Easy-Titer Human IgG Assay Kit. For in vivo studies, groups of 6–8 weeks old mice received IDLV either by intranasal (in) or intramuscular (im) route. mAb production was detected by western blot and ELISA. Mice were challenged using the recombinant IAV VNH5N1-PR8/CDC-RG derived from IAV A/Vietnam/1203/2004.ResultsWe engineered IDLV producing the humanized mAb VN04-2 (IDLV-VN4-2), which is broadly neutralizing against H5 IAV. We found that after transduction of 293T cell with different dosages IDLV-VN4-2, the production of mAb was time and dose dependent. mAb were also functional, and bind specifically H5 HA but not other IAV proteins. We also measured VN04-2 production in the serum of mice 3, 6, 9, 14, 21 and 30 days after in or im administration of IDLV-VN4-2. We found that levels of mAb were sustained. In separate experiments 5/5 mice receiving IDLV-VN4-2 by the in route and 2/5 mice receiving it by the im route were protected from lethal IAV challenge.ConclusionOur data suggest that IDLV may represent an attractive candidate for vector-mediated immunization against infectious disease.Disclosures All authors: No reported disclosures.

HIV-1 REVERSE TRANSCRIPTASE AND PROTEASE ASSAY OF METHANOLIC EXTRACTS OF ADANSONIA DIGITATA L.

&lt;p&gt;&lt;strong&gt;Objective&lt;/strong&gt;:&lt;strong&gt; &lt;/strong&gt;Anti-HIV activity studies of methanolic extracts of &lt;em&gt;Adansonia digitata&lt;/em&gt; L. leaves, root bark and fruit pulp.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods&lt;/strong&gt;:&lt;strong&gt; &lt;/strong&gt;Methanolic extracts of &lt;em&gt;A. digitata&lt;/em&gt; were tested for HIV enzyme inhibitory activity against HIV-1 reverse transcriptase (RT) and HIV-1 protease (PR). HIV-1 RT assay was performed using&lt;em&gt; &lt;/em&gt;non-radioactive HIV-RT colorimetric ELISA kit while the HIV-1 PR assay was performed using a fluorogenic octapeptide substrate, HIV-FRET (fluorescence resonance energy transfer) and a recombinant HIV-1 protease solution (AnaSpec Inc., USA).&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results&lt;/strong&gt;:&lt;strong&gt; &lt;/strong&gt;The percentage inhibition of controls and &lt;em&gt;A. digitata &lt;/em&gt;leaves, root bark and fruit pulp extracts were calculated relative to uninhibited HIV-1 RT and PR in 2% DMSO. The results of the HIV-1 RT assay indicated 26.5% inhibition by root bark extract (50 µg/ml) and 12.2% inhibition by leaf extract while the fruit pulp extract (100 µg/ml) has shown only 5.9% inhibition as compared to standard Nevirapine (100 nM) 27.5% inhibition.&lt;/p&gt;&lt;p&gt;In the case of HIV-protease assay, leaf extract and fruit pulp extract (50 µg/ml) has shown quite high inhibition (≥ 50%) to the extent of 75% and 74% respectively which is very significant. The root bark extract has shown 35% inhibition.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion: &lt;/strong&gt;Methanolic extract of the leaves, root bark and fruit pulp of &lt;em&gt;A. digitata&lt;/em&gt; has shown low anti-HIV-1 RT but significant PR activity. HIV-1 RT activity of root bark extract and HIV-1 PR activity of fruit pulp extract indicates the potential of the plant as an anti-HIV agent. &lt;/p&gt;

707. Attenuated HIV Does Not Amplify in Primary Human T-Lymphocytes During a Model Ex Vivo Gene Therapy Procedure

Current regulatory guidelines require RCL testing of lentiviral vector preparations used in human clinical trials. Cells transduced ex vivo and cultured four days or more must also be tested before administering to the patient. Attenuated HIV (R8.71) can be considered as a theoretical RCL that could develop from a recombination event with wild-type HIV. Importantly, R8.71 lacks accessory proteins (vip, vif, vpr and nef) that are known to be essential for viral replication in T-lymphocytes. Based on this fact, the risk of a recombinant RCL being amplified in T-lymphocytes during an ex vivo transduction and expansion protocol is questionable. To test this possibility, we transduced human T-lymphocytes with one of two RCL-negative lentiviral vector preparations (eGFP or a chimeric antigen receptor (CAR)) that were spiked or not with R8.71 or wild-type HIV. C8166 cells served as a positive control for cells permissive to R8.71. The transduction and subsequent expansion lasted 10 days total and the experiment was performed on two independent occasions. Using day 10 supernatants from all groups, a full RCL assay (with amplification and indicator phases) was performed. Product enhanced reverse transcriptase (PERT) and P24 ELISA assays served as readouts for the RCL assay. Transduction rates were determined by flow cytometry and found to be 57-60% and 10-37% for the eGFP and CAR vector, respectively. The RCL assay results showed that all T-lymphocyte groups exposed to R8.71 were negative but those exposed to wild-type HIV were positive. C8166 cells exposed to R8.71 or wild-type HIV were positive in the RCL assay. These results suggest that a recombinant RCL that possess the wild-type HIV envelope would NOT amplify in human T-lymphocytes during a model ex vivo transduction procedure. The amplification of an arguably more dangerous VSVG-enveloped recombinant RCL is still possible; however, it could be reliably detected by quicker and less expensive assays such as qPCR to VSVG sequences.

The Role of Semen‐Derived Exosomes on Acute HIV Infection in T Lymphocytes

About 35 million people are infected with HIV and even with antiretroviral therapy, the infected individuals are still not living a normal life expectancy. Exosomes, nanovesicles with host proteins and small RNAs, have been shown to possess antiviral properties including suppression of HIV infection. However, these studies were performed in a short‐term setting within 24–48 hours post infection. Here we sought to determine the role of semen exosomes on HIV infection in a longer setting, up to ten days post infection. Semen exosomes were added to acutely infected Jurkat cells and aliquots of the cells and media were taken every two days. The samples were then analyzed for viral integration, replication and fitness either by qPCR, gel electrophoresis or reverse transcriptase assays. Based on the results, exosomes decrease HIV integration, replication and progeny fitness.Support or Funding InformationNSF REU in Microbiology at The University of Iowa through grant number DBI‐1262222

Biochemical Approaches to Study LINE-1 Reverse Transcriptase Activity In Vitro.

In vitro reverse transcriptase assays have been developed to monitor the presence and activity of ORF2p, an essential protein product of the LINE-1 retrotransposon (L1), in cellular fractions. We describe methods for expression and isolation of L1 ribonucleoprotein particles, and identification of ORF2p reverse transcriptase activity. Two independent methods are described: L1 element amplification protocol (LEAP) and direct L1 extension assay (DLEA). The first method involves cDNA synthesis by primer extension using dNTPs followed by a step of PCR amplification. The second method involves primer extension by incorporation of radiolabeled dTMPs followed by dot-blot or gel separation detection. Finally, we discuss the output and benefits of the two methods.

Natural Plant Alkaloid (Emetine) Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity.

Ipecac alkaloids are secondary metabolites produced in the medicinal plant Psychotria ipecacuanha. Emetine is the main alkaloid of ipecac and one of the active compounds in syrup of Ipecac with emetic property. Here we evaluated emetine’s potential as an antiviral agent against Human Immunodeficiency Virus. We performed in vitro Reverse Transcriptase (RT) Assay and Natural Endogenous Reverse Transcriptase Activity Assay (NERT) to evaluate HIV RT inhibition. Emetine molecular docking on HIV-1 RT was also analyzed. Phenotypic assays were performed in non-lymphocytic and in Peripheral Blood Mononuclear Cells (PBMC) with HIV-1 wild-type and HIV-harboring RT-resistant mutation to Nucleoside Reverse Transcriptase Inhibitors (M184V). Our results showed that HIV-1 RT was blocked in the presence of emetine in both models: in vitro reactions with isolated HIV-1 RT and intravirion, measured by NERT. Emetine revealed a strong potential of inhibiting HIV-1 replication in both cellular models, reaching 80% of reduction in HIV-1 infection, with low cytotoxic effect. Emetine also blocked HIV-1 infection of RT M184V mutant. These results suggest that emetine is able to penetrate in intact HIV particles, and bind and block reverse transcription reaction, suggesting that it can be used as anti-HIV microbicide. Taken together, our findings provide additional pharmacological information on the potential therapeutic effects of emetine.

Gammaretrovirus-specific antibodies in free-ranging and captive Namibian cheetahs.

The cheetah population in Namibia is the largest free-ranging population in the world and a key population for research regarding the health status of this species. We used serological methods and quantitative real-time PCR to test free-ranging and captive Namibian cheetahs for the presence of feline leukemia virus (FeLV), a gammaretrovirus that can be highly aggressive in populations with low genetic diversity, such as cheetahs. We also assessed the presence of antibodies to other gammaretroviruses and the responses to a FeLV vaccine developed for domestic cats. Up to 19% of the free-ranging cheetahs, 27% of the captive nonvaccinated cheetahs, and 86% of the captive vaccinated cheetahs tested positive for FeLV antibodies. FeLV-antibody-positive free-ranging cheetahs also tested positive for Rauscher murine leukemia virus antibodies. Nevertheless, FeLV was not detectable by quantitative real-time PCR and no reverse transcriptase activity was detectable by product-enhanced reverse transcriptase assay in the plasma of cheetahs or the supernatants from cultures of peripheral blood mononuclear cells. The presence of antibodies to gammaretroviruses in clinically healthy specimens may be caused either by infection with a low-pathogenic retrovirus or by the expression of endogenous retroviral sequences. The strong humoral immune responses to FeLV vaccination demonstrate that cheetahs can respond to the vaccine and that vaccination against FeLV infection may be beneficial should FeLV infection ever become a threat, as was seen in Iberian lynx and Florida panthers.

Detection of Human Metapneumovirus in Hospitalized Children with Acute Respiratory Tract Infections in Sulaimani Province, Iraq

Background: Human Metapneumovirus (hMPV) is a member of Paramyxoviridae family. It is an important viral pathogen of respiratory tract infections among children. Therefore, the aim of this study is to determine the incidence of hMPV infections among children less than 5 years of age with respiratory tract infections at the pediatric hospital in Sulaimani city of Iraq. Also, this study aims to evaluate the different diagnostic methods for the detection of this virus. Method: Nasopharyngeal swabs and throat swabs were collected from 300 hospitalized children with respiratory infections under 5 years of age between April 2011 and March 2012. Each sample was used for hMPV detection by conventional reverse transcriptase (RT-PCR) and direct fluorescent assay (DFA). A questionnaire which was designed for acute respiratory tract infections among hospitalized children was also included. Results: Human Metapneumovirus was detected in 16% by RT-PCR and 14.7% by DFA. Autumn – winter period was the most common season for hMPV infections with its peak occurrence in January and February. Conclusions: hMPV is an important pathogen associated with RTIs in children. RT-PCR is highly sensitive and specific for the detection of hMPV than antigen detection methods used for the diagnosis of hMPV viruses.