Viral Ribonucleic Acid Research Articles

Evaluation of a commercially available synthetic RNA lipid enveloped control molecule

Detection of viral ribonucleic acid with RT PCR is a useful tool for viral detection. One of the drawbacks of this technique is the difficulty in including an internal control molecule to ensure the validity of the extraction and amplification process. In this study the potential usefulness of a novel lipid enveloped commercially available RNA control molecule is investigated.Initial optimisation of the detection assay was performed by amplification of IC (internal control) spiked into PCR water. Thirty-two clinical respiratory samples were spiked with the IC before and after extraction and RT PCR was then performed. Inefficient extraction was simulated. Inhibition of the RT PCR was achieved by serial dilution of heparin sulfate into samples post extraction.No Targets that matched the IC (Internal Control) primers were identified in 32 extracted sputum samples as determined by the absence of non specific amplification curves. The unextracted IC had an increased CT (cycle threshold) value compared to IC that had been extracted. Inefficient extraction was detected by an increased CT. Increasing concentrations of heparin inhibited the PCR in a predictable fashion.The Bioline IC molecule provides a stable RNA IC that has acceptable performance characteristics.

Commentary

Commentary

Detection of peste des petits ruminants virus in formalin-fixed tissues.

Peste des petits ruminants virus that causes a highly infectious and often fatal disease of sheep and goats is confirmed by various diagnostic techniques among them being isolation of the virus from cell culture systems, viral ribonucleic acid (RNA) detection by molecular assays, and viral antigen detection by immunocapture enzyme-linked immunosorbent assay (IC ELISA), immunohistochemistry (IHC), and AGAR gel test. Whereas most of the confirmatory diagnostic procedures require pathological samples to be stored frozen to preserve integrity of the peste des petits ruminants (PPR) virus RNA, samples for IHC tests are preserved in 10% formalin. In this study, nine formalin-fixed pathological samples from three goats suspected of PPR were processed for extraction of PPR viral RNA and analyzed for detection with real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay. The results showed that five out of the nine tested samples returned positive for presences PPR viral genome. This study has established that field pathological samples of PPR-suspected cases, collected and stored in 10% formalin for up 2 years, could be used for PPR virus RNA extraction for disease virus confirmation.

Being prepared: bioterrorism and mass prophylaxis: part II.

Although several biological agents have been recognized as presenting a significant threat to public health if used in a bioterrorist attack, those that are of greatest importance are known as the Category A agents: Bacillus anthracis (anthrax); variola major (smallpox); Yersinia pestis (plague); Francisella tularensis (tularemia); ribonucleic acid viruses (hemorrhagic fevers); and Clostridium botulinum (botulism toxin). In the previous issue, Part I of this review focused on the clinical presentation and treatment of anthrax, plague, and tularemia. In this second part of this 2-part review of these agents, the focus is on the clinical presentation and treatment of smallpox, viral hemorrhagic fevers, and botulism toxin. The utilization of mass prophylaxis to limit the morbidity and mortality associated with all these agents is also discussed along with the role emergency care personnel play in its implementation.

A microfluidic system integrated with buried optical fibers for detection of Phalaenopsis orchid pathogens

Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Fast, accurate, and on-site detection of pathogens in these orchids is therefore of critical importance in order to prevent or suppress costly disease outbreaks. Traditional pathogen detection methods are time-consuming, require well-equipped laboratories with highly trained personnel, and cannot be conducted in situ. In this study, a microfluidic system integrated with buried optical fibers was developed to detect viral pathogens of Phalaenopsis spp. Briefly, virus-specific ribonucleic acid (RNA) purification was achieved by a pre-treatment incubation with magnetic beads, and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was used subsequently to amplify the viral RNA. Positive RT-LAMP reactions resulted in the precipitation of magnesium pyrophosphate, which caused a change in turbidity that could be seen by the naked eye. A buried optical fiber-based detection module and a micro-stirring device were then integrated into the microfluidic chip to detect the RT-LAMP reaction product directly on the chip itself by measuring the change in the optical signals caused by the turbidity change associated with a positive amplification. The limit of detection for this system was found to be 25fg, which is of similar sensitivity to existing, more laborious methods. Therefore, by using the integrated microfluidic system, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65min.

Reovirus in cancer therapy: an evidence-based review

Reovirus, a double-stranded ribonucleic acid virus and benign human pathogen, preferentially infects and kills cancer cells in its unmodified form, and is one of the leading oncolytic viruses currently undergoing clinical trials internationally. With 32 clinical trials completed or ongoing thus far, reovirus has demonstrated clinical therapeutic applicability against a multitude of cancers, including but not limited to breast cancer, prostate cancer, pancreatic cancer, malignant gliomas, advanced head and neck cancers, and metastatic ovarian cancers. Phase I trials have demonstrated that reovirus is safe to use via both intralesional/intratumoral and systemic routes of administration, with the most common adverse reactions being grade I/II toxicities, such as flu-like illness (fatigue, nausea, vomiting, headache, fever/chills, dizziness), diarrhea, and lymphopenia. In subsequent Phase II trials, reovirus administration was demonstrated to successfully decrease tumor size and promote tumor necrosis, thereby complementing compelling preclinical evidence of tumor destruction by the virus. Importantly, reovirus has been shown to be effective as a monotherapy, as well as in combination with other anticancer options, including radiation and chemotherapeutic agents, such as gemcitabine, docetaxel, paclitaxel, and carboplatin. Of note, the first Phase III clinical trial using reovirus in combination with paclitaxel and carboplatin for the treatment of head and neck cancers is under way. Based on the evidence from clinical trials, we comprehensively review the use of reovirus as an anticancer agent, acknowledge key obstacles, and suggest future directions to ultimately potentiate the efficacy of reovirus oncotherapy.

Prevalence of HCV infection and associated factors among illicit drug users in Breves, State of Pará, northern Brazil.

Illicit drug users (DUs) are vulnerable to hepatitis C virus (HCV) infection. The shared use of illicit drugs is the main method of HCV transmission. A cross-sectional study was conducted in Breves, in northern Brazil. We surveyed 187 DUs to determine the prevalence of and factors associated with HCV infection. The prevalence of anti-HCV antibodies was 36.9%, and the prevalence of hepatitis C virus-ribonucleic acid (HCV-RNA) was 31%. Hepatitis C virus infection was associated with tattoos, intravenous drug use, shared use of equipment for drug use, drug use for longer than 3 years, and daily drug use. Strategies for preventing and controlling HCV transmission should be implemented among DUs.

Complications infectieuses chez le transplanté hépatique

Infectious complications after liver transplantation are common and may be life-threatening in the short and even long terms. The risk is related to the underlying endstage liver disease, major abdominal surgery, and immunosuppressive therapy administered in the postoperative period and further for the whole patient’s life. Bacterial infections are dominated by intra-abdominal infections, which mostly start by biliary and vascular surgical complications as well as respiratory and bloodstream infections, especially during the first six months. The risk of invasive fungal infection is particularly high. Targeted or universal prophylaxis or preemptive measures have significantly reduced the risk of several infections including Candida, P. jirovecii, and cytomegalovirus infections. In contrast, diagnosis of invasive aspergillosis remains difficult and its subsequent mortality high. In hepatitis C virus (HCV) ribonucleic acid carriers, the systematic HCV recurrence on the graft after liver transplantation in the absence of prior therapy is associated with cirrhosis development and long-term poor outcome. It represents the most common long-term infectious complication. Efficiency of the available treatments is limited due to the low rate of sustained viral response and poor tolerance. The perspective of direct HCV antivirals including inhibitors of protease, polymerase, and other non-structural HCV proteins represents a major step in the treatment of HCV recurrence after liver transplantation.

Do differences exist between chronic hepatitis C genotypes 2 and 3?

Six genotypes of the hepatitis C virus (HCV) have been identified thus far, and their distribution is well defined. Genotype 1, which is the most prevalent worldwide, is always compared to genotypes 2 and 3, particularly in terms of treatment response. However, little is known about the differences between genotypes 2 and 3 because these genotypes are analyzed together in most studies. Therefore, the aim of this study was to evaluate differences in the clinical, epidemiological, laboratory, and histological parameters between HCV-2 and HCV-3. Patients with chronic hepatitis C infected with genotypes 2 and 3 were studied retrospectively and compared according to clinical, laboratory, and histological aspects. Hepatitis C virus-ribonucleic acid (HCV-RNA) was analyzed quantitatively by TaqMan® real-time PCR, and the HCV genotype was determined by sequencing the 5'-untranslated region. A total of 306 patients with chronic HCV-2 (n=50) and HCV-3 (n = 256) were studied. Subtype 2b (n=17/50) and subtype 3a (n=244/256) were the most prevalent among patients infected with HCV-2 and HCV-3, respectively. The mean age was 47 ± 10 years, and there was a predominance of men in the group studied (61%). Comparative analysis between HCV-2 and HCV-3 showed a younger age (p=0.002), less prevalence of arterial hypertension (p=0.03), higher serum albumin levels (p=0.01), more advanced stage of liver fibrosis (p=0.03), and higher frequency of steatosis in patients with HCV-3 (p=0.001). After multivariate regression analysis, all the variables, except serum albumin, remained as variables associated with HCV-3 in the final model. Clinical and histological differences exist between HCV-2 and HVC-3, which suggests the need for separate analyses of these genotypes.

Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein

Toscana virus is an emerging bunyavirus in Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negative-strand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric single-stranded RNA. RNA is sequestered in a sequence-independent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses.

Severe thrombocytopenia in a patient with inosine triphosphatase (ITPA)–CC genotype caused by pegylated interferon (IFN)-α-2a with ribavirin therapy: a case report

BackgroundPegylated interferon combined with ribavirin treatment is an effective therapy for chronic hepatitis C viral infection. However, pegylated interferon combined with ribavirin is associated with various adverse reactions. Severe thrombocytopenia is a life-threatening side effect of interferon therapy that can lead to bleeding. It is generally understood that the inosine triphosphatase-CC genotype does not have a significantly lower reduction by pegylated interferon combined with ribavirin in the mean platelet counts compared with the AA/CA genotype. We report a case of severe thrombocytopenia that developed in a patient with chronic hepatitis C treated with pegylated interferon combined with ribavirin in spite of having the inosine triphosphatase-CC genotype.Case presentationA 57-year-old female had been diagnosed as having HCV infection in 2008. The inosine triphosphatase gene showed one single nucleotide polymorphism (rs1127354) C/C (major homozygous) and the IL28B gene showed single nucleotide polymorphism (rs8099917 T/T, rs11881222 T/T) (major homozygous). The patient was treated with pegylated interferon 180 μg once a week combined with ribavirin 600 mg per day from April 2011. The hepatitis c virus ribonucleic acid turned negative 9 weeks after treatment with pegylated interferon combined with ribavirin. During the therapy, the platelet count remained above 8.0 × 104/μl for about 9 months. In January 2012, the platelet count was 6.8 × 104/μl. In February 2012, the 44th week from the beginning of the treatment, a sudden decrease in the platelet count to 0.8 × 104/μl was observed. After prednisolone was administered, the platelet count increased. Finally the platelet count had risen above normal range.ConclusionWe should pay careful attention in the differential diagnosis for patients with the inosine triphosphatase-CC genotype because, although rare, severe thrombocytopenia could occur.

Association between histological findings, aminotransferase levels and viral genotype in chronic hepatitis C infection

The genomic heterogeneity of hepatitis C virus (HCV) influences liver disorders. This study aimed to determine the prevalence of HCV genotypes and to investigate the influence of these genotypes on disease progression. Blood samples and liver biopsies were collected from HCV-seropositive patients for serological analysis, biochemical marker measurements, HCV genotyping and histopathological evaluation. Hepatitis C virus-ribonucleic acid (HCV-RNA) was detected in 107 patients (90.6% with genotype 1 and 9.4% with genotype 3). Patients infected with genotype 1 exhibited higher mean necroinflammatory activity and fibrosis. HCV genotype 1 was the most prevalent and was associated with greater liver dysfunction.

Dynamic and geometric analyses of Nudaurelia capensis ω virus maturation reveal the energy landscape of particle transitions

Quasi-equivalent viruses that infect animals and bacteria require a maturation process in which particles transition from initially assembled procapsids to infectious virions. Nudaurelia capensis ω virus (NωV) is a T = 4, eukaryotic, single-stranded ribonucleic acid virus that has proved to be an excellent model system for studying the mechanisms of viral maturation. Structures of NωV procapsids (diameter = 480 Å), a maturation intermediate (410 Å), and the mature virion (410 Å) were determined by electron cryo-microscopy and three-dimensional image reconstruction (cryoEM). The cryoEM density for each particle type was analyzed with a recently developed maximum likelihood variance (MLV) method for characterizing microstates occupied in the ensemble of particles used for the reconstructions. The procapsid and the mature capsid had overall low variance (i.e., uniform particle populations) while the maturation intermediate (that had not undergone post-assembly autocatalytic cleavage) had roughly two to four times the variance of the first two particles. Without maturation cleavage, the particles assume a variety of microstates, as the frustrated subunits cannot reach a minimum energy configuration. Geometric analyses of subunit coordinates provided a quantitative description of the particle reorganization during maturation. Superposition of the four quasi-equivalent subunits in the procapsid had an average root mean square deviation (RMSD) of 3 Å while the mature particle had an RMSD of 11 Å, showing that the subunits differentiate from near equivalent environments in the procapsid to strikingly non-equivalent environments during maturation. Autocatalytic cleavage is clearly required for the reorganized mature particle to reach the minimum energy state required for stability and infectivity.

Experimentally Infected Domestic Ducks Show Efficient Transmission of Indonesian H5N1 Highly Pathogenic Avian Influenza Virus, but Lack Persistent Viral Shedding

Ducks are important maintenance hosts for avian influenza, including H5N1 highly pathogenic avian influenza viruses. A previous study indicated that persistence of H5N1 viruses in ducks after the development of humoral immunity may drive viral evolution following immune selection. As H5N1 HPAI is endemic in Indonesia, this mechanism may be important in understanding H5N1 evolution in that region. To determine the capability of domestic ducks to maintain prolonged shedding of Indonesian clade 2.1 H5N1 virus, two groups of Pekin ducks were inoculated through the eyes, nostrils and oropharynx and viral shedding and transmission investigated. Inoculated ducks (n = 15), which were mostly asymptomatic, shed infectious virus from the oral route from 1 to 8 days post inoculation, and from the cloacal route from 2–8 dpi. Viral ribonucleic acid was detected from 1–15 days post inoculation from the oral route and 1–24 days post inoculation from the cloacal route (cycle threshold <40). Most ducks seroconverted in a range of serological tests by 15 days post inoculation. Virus was efficiently transmitted during acute infection (5 inoculation-infected to all 5 contact ducks). However, no evidence for transmission, as determined by seroconversion and viral shedding, was found between an inoculation-infected group (n = 10) and contact ducks (n = 9) when the two groups only had contact after 10 days post inoculation. Clinical disease was more frequent and more severe in contact-infected (2 of 5) than inoculation-infected ducks (1 of 15). We conclude that Indonesian clade 2.1 H5N1 highly pathogenic avian influenza virus does not persist in individual ducks after acute infection.

RNA Viruses: ROS-Mediated Cell Death.

Reactive oxygen species (ROS) are well known for being both beneficial and deleterious. The main thrust of this review is to investigate the role of ROS in ribonucleic acid (RNA) virus pathogenesis. Much evidences has accumulated over the past decade, suggesting that patients infected with RNA viruses are under chronic oxidative stress. Changes to the body's antioxidant defense system, in relation to SOD, ascorbic acid, selenium, carotenoids, and glutathione, have been reported in various tissues of RNA-virus infected patients. This review focuses on RNA viruses and retroviruses, giving particular attention to the human influenza virus, Hepatitis c virus (HCV), human immunodeficiency virus (HIV), and the aquatic Betanodavirus. Oxidative stress via RNA virus infections can contribute to several aspects of viral disease pathogenesis including apoptosis, loss of immune function, viral replication, inflammatory response, and loss of body weight. We focus on how ROS production is correlated with host cell death. Moreover, ROS may play an important role as a signal molecule in the regulation of viral replication and organelle function, potentially providing new insights in the prevention and treatment of RNA viruses and retrovirus infections.

The emergence of ebola as a global health security threat: from 'lessons learned' to coordinated multilateral containment efforts.

First reported in remote villages of Africa in the 1970s, the Ebolavirus was originally believed to be transmitted to people from wild animals. Ebolavirus (EBOV) causes a severe, frequently fatal hemorrhagic syndrome in humans. Each outbreak of the Ebolavirus over the last three decades has perpetuated fear and economic turmoil among the local and regional populations in Africa. Until now it has been considered a tragic malady confined largely to the isolated regions of the African continent, but it is no longer so. The frequency of outbreaks has increased since the 1970s. The 2014 Ebola outbreak in Western Africa has been the most severe in history and was declared a public health emergency by the World Health Organization. Given the widespread use of modern transportation and global travel, the EBOV is now a risk to the entire Global Village, with intercontinental transmission only an airplane flight away. Clinically, symptoms typically appear after an incubation period of approximately 11 days. A flu-like syndrome can progress to full hemorrhagic fever with multiorgan failure, and frequently, death. Diagnosis is confirmed by detection of viral antigens or Ribonucleic acid (RNA) in the blood or other body fluids. Although historically the mortality of this infection exceeded 80%, modern medicine and public health measures have been able to lower this figure and reduce the impact of EBOV on individuals and communities. The treatment involves early, aggressive supportive care with rehydration. Core interventions, including contact tracing, preventive initiatives, active surveillance, effective isolation and quarantine procedures, and timely response to patients, are essential for a successful outbreak control. These measures, combined with public health education, point-of-care diagnostics, promising new vaccine and pharmaceutical efforts, and coordinated efforts of the international community, give new hope to the Global effort to eliminate Ebola as a public health threat. Here we present a review of EBOV infection in an effort to further educate medical and political communities on what the Ebolavirus disease entails, and what efforts are recommended to treat, isolate, and eventually eliminate it.

Understanding the Role of RNA in Driving the Clustering of HIV Gag Molecules using Coarse-Grained Molecular Dynamics Models

Multi-domain Gag protein is the fundamental building block of the retrovirus particles. In the process of the HIV-1 particle assembly, Gag poly-protein interacts with lipids, with ribonucleic acids (RNAs) and with other Gag proteins. These diverse interactions drive the construction of the infectious particle and the packaging of the viral RNA into the particle. In this work, we have used coarse-grained (CG) molecular dynamics models of plasma membrane, RNA and Gag poly-protein to understand the role of various components in the formation of virus-like particles (VLPs). In particular, we focus on the possible role that the genomic RNA plays as a scaffolding agent in driving the clustering and assembly of Gag proteins on the membrane. We use available CG models of Gag proteins [Ayton & Voth, BJ 2010] and lipids [Srivastava & Voth, JCTC 2012] and develop a new CG model (one-site per nucleotide) for the full genomic RNA using the experimentally available architecture and secondary structure of the entire HIV-1 NL4-3 genome. [Watts et. Al, Nature 2009; Jonikas et al, RNA 2009] Our simulations confirm that the extended Gag molecules are stable only when they are simultaneously associated with both the membrane and the RNA. Using our model, we also show the aggregation of distributed Gag molecules on the membrane surface in presence of RNA strands. Preliminary data from our simulations indicate that existing membrane-bound RNA-Gag seed complexes act as nucleation sites and possibly assist in capturing the diffusing Gag molecule at a distance on the bilayer, slowly increasing the cluster sizes. Large-scale simulation with full genomic RNA is expected to provide further insights into the scaffolding mechanism.

Enhancement of accuracy and efficiency for RNA secondary structure prediction by sequence segmentation and MapReduce

BackgroundRibonucleic acid (RNA) molecules play important roles in many biological processes including gene expression and regulation. Their secondary structures are crucial for the RNA functionality, and the prediction of the secondary structures is widely studied. Our previous research shows that cutting long sequences into shorter chunks, predicting secondary structures of the chunks independently using thermodynamic methods, and reconstructing the entire secondary structure from the predicted chunk structures can yield better accuracy than predicting the secondary structure using the RNA sequence as a whole. The chunking, prediction, and reconstruction processes can use different methods and parameters, some of which produce more accurate predictions than others. In this paper, we study the prediction accuracy and efficiency of three different chunking methods using seven popular secondary structure prediction programs that apply to two datasets of RNA with known secondary structures, which include both pseudoknotted and non-pseudoknotted sequences, as well as a family of viral genome RNAs whose structures have not been predicted before. Our modularized MapReduce framework based on Hadoop allows us to study the problem in a parallel and robust environment.ResultsOn average, the maximum accuracy retention values are larger than one for our chunking methods and the seven prediction programs over 50 non-pseudoknotted sequences, meaning that the secondary structure predicted using chunking is more similar to the real structure than the secondary structure predicted by using the whole sequence. We observe similar results for the 23 pseudoknotted sequences, except for the NUPACK program using the centered chunking method. The performance analysis for 14 long RNA sequences from the Nodaviridae virus family outlines how the coarse-grained mapping of chunking and predictions in the MapReduce framework exhibits shorter turnaround times for short RNA sequences. However, as the lengths of the RNA sequences increase, the fine-grained mapping can surpass the coarse-grained mapping in performance.ConclusionsBy using our MapReduce framework together with statistical analysis on the accuracy retention results, we observe how the inversion-based chunking methods can outperform predictions using the whole sequence. Our chunk-based approach also enables us to predict secondary structures for very long RNA sequences, which is not feasible with traditional methods alone.

English

Quantitative tests for the detection of hepatitis C virus ribonucleic acid (HCV-RNA) levels and HCV core antigen have been mainly used for the management of patients with HCV infection. The aim of this study was to evaluate the correlation between HCV core antigen and HCV RNA levels in patients reactive for anti-HCV antibodies. Three hundred and twenty-five anti-HCV reactive sera samples were included in the study. HCV core antigen and HCV RNA levels were determined using the Architect HCV Ag test and Abbott RealTimeTM HCV RNA test (RT-PCR), respectively. The correlation coefficient between the levels of HCV core antigen and HCV RNA test results was calculated using Spearman&rsquo;s rank test, and linear regression analysis was applied. One hundred and sixteen of the 325 samples were detected positive by both methods. Three additional samples by RT-PCR, and 4 samples by Architect HCV Ag, the negative samples were found positive by the other method. All of these contradictory results were obtained from the low level HCV RNA or HCV core antigen including samples. A correlation coefficient (r) was determined as 0.899 between the levels of HCV core antigen and HCV RNA (p&lt;0.0001). The sensitivity, specificity, positive predictive value and negative predictive value of the HCV core antigen test were 97.48, 98.06, 96.67 and 98.54%, respectively, using the HCV RNA test as a reference. The Architect HCV core antigen test exhibits a good correlation with the HCV RNA test. It can be used as an alternative method, especially when the HCV RNA test is unavailable. &nbsp; Key words: Anti- hepatitis C virus, Architect hepatitis C virus Ag test, hepatitis C virus RNA.

Hepatobiliary Quiz-7 (2013)

HEV is a single stranded positive sense 7.2 kilo base pair ribonucleic acid (RNA) virus consisting of three overlapping open reading frames ORF1, ORF2 and ORF3.1 The largest of these, ORF1 forms the 5′ distal end of the genome and codes for the non-structural replication proteins including the RNA dependent RNA polymerase (RdRp). The 3′ ORF2 codes for an arginine rich major structural protein.2-4 ORF3 codes for a small protein thought to be a minor structural protein which has a role in viral egress and association into virons.