Viral RNA Target Research Articles

The Induction of an Effective dsRNA-Mediated Resistance Against Tomato Spotted Wilt Virus by Exogenous Application of Double-Stranded RNA Largely Depends on the Selection of the Viral RNA Target Region.

Tomato spotted wilt virus (TSWV) is a devastating plant pathogen, causing huge crop losses worldwide. Unfortunately, due to its wide host range and emergence of resistance breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference (RNAi) has been achieved only in transgenic plants expressing parts of the viral genome or artificial microRNAs targeting it. Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based vaccination, represents an attractive and promising alternative, already shown to be effective against different positive-sense RNA viruses and viroids. In the present study, the protection efficacy of exogenous application of dsRNAs targeting the nucleocapsid (N) or the movement protein (NSm) coding genes of the negative-sense RNA virus TSWV was evaluated in Nicotiana benthamiana as model plant and in tomato as economically important crop. Most of the plants treated with N-targeting dsRNAs, but not with NSm-targeting dsRNAs, remained asymptomatic until 40 (N. benthamiana) and 63 (tomato) dpi, while the remaining ones showed a significant delay in systemic symptoms appearance. The different efficacy of N- and NSm-targeting dsRNAs in protecting plants is discussed in the light of their processing, mobility and biological role. These results indicate that the RNAi-based vaccination is effective also against negative-sense RNA viruses but emphasize that the choice of the target viral sequence in designing RNAi-based vaccines is crucial for its success.

Non-retroviral Endogenous Viral Element Limits Cognate Virus Replication in Aedes aegypti Ovaries

SummaryEndogenous viral elements (EVEs) are viral sequences integrated in host genomes. A large number of non-retroviral EVEs was recently detected in Aedes mosquito genomes, leading to the hypothesis that mosquito EVEs may control exogenous infections by closely related viruses. Here, we experimentally investigated the role of an EVE naturally found in Aedes aegypti populations and derived from the widespread insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR-Cas9 genome editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE resulted in increased CFAV replication in ovaries, possibly modulating vertical transmission of the virus. Viral replication was controlled by targeting of viral RNA by EVE-derived P-element-induced wimpy testis-interacting RNAs (piRNAs). Our results provide evidence that antiviral piRNAs are produced in the presence of a naturally occurring EVE and its cognate virus, demonstrating a functional link between non-retroviral EVEs and antiviral immunity in a natural insect-virus interaction.

Molecular Mechanism of RNA Recognition by Zinc-Finger Antiviral Protein

Zinc-finger antiviral protein (ZAP) is a host antiviral factor that specifically restricts a wide range of viruses. ZAP selectively binds to CG-dinucleotide-enriched RNA sequences and recruits multiple RNA degradation machines to degrade target viral RNA. However, the molecular mechanism and structural basis for ZAP recognition of specific RNA are not clear. Here, we report the crystal structure of the ZAP N-terminal domain bound to a CG-rich single-stranded RNA, providing the molecular basis for its specific recognition of a CG dinucleotide and additional guanine and cytosine. The four zinc fingers of ZAP adopt a unique architecture and form extensive interactions with RNA. Mutations of both protein and RNA at the RNA-ZAP interacting surface reduce the invitro binding affinity and cellular antiviral activity. This work reveals the molecular mechanism of ZAP recognition of specific target RNA and also provides insights into the mechanism by which ZAP coordinates downstream RNA degradation processes.

Label-free fluorometric detection of influenza viral RNA by strand displacement coupled with rolling circle amplification.

Since influenza occurs globally every year, it is important to develop a facile and accurate method to detect the influenza virus. This study aimed at developing a sensitive fluorometric assay for detecting influenza viral RNA through tandem gene amplification methods including reverse transcription PCR (RT-PCR), followed by strand displacement amplification (SDA) coupled with rolling circle amplification (RCA). Influenza viral RNA was initially amplified by RT-PCR with a tailed reverse primer containing an additional sequence for SDA. The RT-PCR amplicon was then subjected to SDA, yielding multiple copies of single-stranded DNA (ssDNA) that can be used as a primer for subsequent RCA. Thereafter, a long ssDNA segment harboring tandem repeated G-quadruplexes that were generated through RCA was intercalated by Thioflavin T, yielding a strong fluorescence signal indicating the presence of the target viral RNA. Fluorometric analysis detected influenza viral RNA ranging from 50 pg to 500 pg with a limit of detection of 6.2 pg with a signal-to-background ratio of 10 and identified each influenza virus strain (H1N1, H3N2, and influenza B). Thus, the present method for the label-free fluorometric detection of viral RNA via tandem gene amplifications combining RT-PCR-coupled SDA and G-quadruplex-generating RCA would facilitate the efficient diagnosis of influenza infection.

Preliminary evaluation of Thermo Fisher TaqMan® Triplex q-PCR kit for simultaneous detection of chikungunya, dengue, and Zika viruses in mosquitoes.

Preliminary evaluation of Thermo Fisher TaqMan® Triplex q-PCR kit for simultaneous detection of chikungunya, dengue, and Zika viruses in mosquitoes.

Plant Translation Initiation Complex eIFiso4F Directs Pokeweed Antiviral Protein to Selectively Depurinate Uncapped Tobacco Etch Virus RNA.

Pokeweed antiviral protein (PAP) is a ribosome inactivating protein (RIP) that depurinates the sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. PAP depurinates viral RNA, and in doing so, lowers the infectivity of many plant viruses. The mechanism by which PAP accesses uncapped viral RNA is not known, impeding scientists from developing effective antiviral agents for the prevention of the diseases caused by uncapped RNA viruses. Kinetic rates of PAP interacting with tobacco etch virus (TEV) RNA, in the presence and absence of eIFiso4F, were examined, addressing how the eIF affects selective PAP targeting and depurination of the uncapped viral RNA. PAP-eIFs copurification assay and fluorescence resonance energy transfer demonstrate that PAP forms a ternary complex with the eIFiso4G and eIFiso4E, directing the depurination of uncapped viral RNA. eIFiso4F selectively targets PAP to depurinate TEV RNA by increasing PAP's specificity constant for uncapped viral RNA 12-fold, when compared to the depurination of an oligonucleotide RNA that mimics the SRL of large rRNA, and cellular capped luciferase mRNA. This explains how PAP is able to lower infectivity of pokeweed viruses, while preserving its own ribosomes and cellular RNA from depurination: PAP utilizes cellular eIFiso4F in a novel strategy to target uncapped viral RNA. It may be possible to modulate and utilize these PAP-eIFs interactions for their public health benefit; by repurposing them to selectively target PAP to depurinate uncapped viral RNA, many plant and animal diseases caused by these viruses could be alleviated.

Fluorometric detection of influenza virus RNA by PCR-coupled rolling circle amplification generating G-quadruplex

We developed a label-free fluorometric system based on double amplification of target nucleic acid by rolling circle amplification generating G-quadruplex (GQ-RCA) for sensitive detection of influenza virus RNA. To detect viral RNA with high sensitivity and selectivity, double amplification system was employed through reverse transcription PCR (RT-PCR) coupled in tandem with GQ-RCA. Single-stranded amplified viral DNA (ssDNA) was generated by RT-PCR of samples containing influenza virus RNA. The phosphate-primed DNA strand in amplicon DNA was subsequently degraded by lambda exonuclease. Viral ssDNA formed a ternary RCA initiation complex by annealing to both a partial hairpin primer and a dumbbell padlock DNA template. A long stretch of ssDNA containing repeated copies of the G-quadruplex sequence was generated by RCA at room temperature. Sensitive detection of amplified target nucleic acid was then accomplished by monitoring fluorogenic interactions between Thioflavin T (ThT) and RCA-responsive G-quadruplexes. Differences in fluorescence intensity between target and non-target viral RNA were evident under UV illumination. Selective fluorescence staining of RCA-responsive G-quadruplexes enabled influenza virus RNA detection at concentrations as low as 4.9aM with a linear detection range between 450aM and 450fM. The RT-PCR-coupled GQ-RCA system for influenza virus genome detection exhibited very high sensitivity and allowed convenient multiplexed virus detection within 2.5h. Thus, a combination of RT-PCR-coupled GQ-RCA and ThT fluorescence staining can be a sensitive and accurate method for detecting RNA molecules of influenza viruses as well as those of other viruses.

Translational control in plant antiviral immunity.

Due to the limited coding capacity of viral genomes, plant viruses depend extensively on the host cell machinery to support the viral life cycle and, thereby, interact with a large number of host proteins during infection. Within this context, as plant viruses do not harbor translation-required components, they have developed several strategies to subvert the host protein synthesis machinery to produce rapidly and efficiently the viral proteins. As a countermeasure against infection, plants have evolved defense mechanisms that impair viral infections. Among them, the host-mediated translational suppression has been characterized as an efficient mean to restrict infection. To specifically suppress translation of viral mRNAs, plants can deploy susceptible recessive resistance genes, which encode translation initiation factors from the eIF4E and eIF4G family and are required for viral mRNA translation and multiplication. Additionally, recent evidence has demonstrated that, alternatively to the cleavage of viral RNA targets, host cells can suppress viral protein translation to silence viral RNA. Finally, a novel strategy of plant antiviral defense based on suppression of host global translation, which is mediated by the transmembrane immune receptor NIK1 (nuclear shuttle protein (NSP)-Interacting Kinase1), is discussed in this review.

Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, ‘Strand Invasion Based Amplification’ (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7491-y) contains supplementary material, which is available to authorized users.

A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics.

The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR) and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR), thus expanding the TTC’s ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC’s performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings.

Discovery of Influenza A Virus Sequence Pairs and Their Combinations for Simultaneous Heterosubtypic Targeting that Hedge against Antiviral Resistance.

The multiple circulating human influenza A virus subtypes coupled with the perpetual genomic mutations and segment reassortment events challenge the development of effective therapeutics. The capacity to drug most RNAs motivates the investigation on viral RNA targets. 123,060 segment sequences from 35,938 strains of the most prevalent subtypes also infecting humans–H1N1, 2009 pandemic H1N1, H3N2, H5N1 and H7N9, were used to identify 1,183 conserved RNA target sequences (≥15-mer) in the internal segments. 100% theoretical coverage in simultaneous heterosubtypic targeting is achieved by pairing specific sequences from the same segment (“Duals”) or from two segments (“Doubles”); 1,662 Duals and 28,463 Doubles identified. By combining specific Duals and/or Doubles to form a target graph wherein an edge connecting two vertices (target sequences) represents a Dual or Double, it is possible to hedge against antiviral resistance besides maintaining 100% heterosubtypic coverage. To evaluate the hedging potential, we define the hedge-factor as the minimum number of resistant target sequences that will render the graph to become resistant i.e. eliminate all the edges therein; a target sequence or a graph is considered resistant when it cannot achieve 100% heterosubtypic coverage. In an n-vertices graph (n ≥ 3), the hedge-factor is maximal (= n– 1) when it is a complete graph i.e. every distinct pair in a graph is either a Dual or Double. Computational analyses uncover an extensive number of complete graphs of different sizes. Monte Carlo simulations show that the mutation counts and time elapsed for a target graph to become resistant increase with the hedge-factor. Incidentally, target sequences which were reported to reduce virus titre in experiments are included in our target graphs. The identity of target sequence pairs for heterosubtypic targeting and their combinations for hedging antiviral resistance are useful toolkits to construct target graphs for different therapeutic objectives.

Next-Generation Sequencing Confirmation of Real-Time RT-PCR False Positive Influenza-A Virus Detection in Waterfowl and Swine Swab Samples

Surveillance for the Influenza A virus is the most important method used to monitor poultry and other animal species for the presence of the Influenza A viruses. Waterfowl and swine swab samples that tested positive for the Influenza A virus by real-time RT-PCR (rRT-PCR) but tested negative for the virus by virus isolation were further investigated in our recent studies using next-generation sequencing (NGS). A total of seven such pooled swab samples (four swine oral-pharyngeal (OPH) swab pools and three wild duck OPH and cloacal swab pools) were tested for influenza A virus by whole genome sequencing using NGS with the Illumina MiSeq system. Our sequencing results confirmed that none of these rRT-PCR positive samples (Ct-values of 22 to 28) contained Influenza A virus contigs; instead, all the samples contained multiple non-target contig sequences mismatched to the rRT-PCR primer and probe sequences, which led to the positive rRT-PCR results. These genome sequence findings provide scientific evidence that the rRT-PCR false positive results were caused by non-target contigs and not by target viral RNA. Additionally, these samples tested negative for the influenza A virus by conventional RT-PCR (cRT-PCR), which suggests cRT-PCR can serve as an alternative approach for identifying rRT-PCR false positive results.

Poliovirus and Group C Enteroviruses: Knowledge Gaps Relevant to Eradication

Poliovirus eradication is one of the most challenging public health endeavors in modern times (www.polioeradication.org). Social, political, economic & scientific factors have made this goal elusive. When eradication goals were first established in 1988, there was little appreciation of viral RNA recombination, enterovirus species groups and their relevance to eradication. Now, it is clear that RNA recombination between live-attenuated vaccine strains of poliovirus and non-polio group C enteroviruses results in circulating vaccine-derived polioviruses (cVDPV) and corresponding outbreaks of paralytic disease, a significant obstacle to eradication. By understanding enterovirus species groups, it becomes clear that poliovirus capsid proteins can be eradicated; however, the remainder of poliovirus RNA genomes will survive indefinitely in other group C enteroviruses. To help address these obstacles to eradication, the Barton lab studies molecular features of 3D pol involved in viral RNA replication and recombination. A dsRNA clamp of 3D pol that holds RNA products of replication as they exit the polymerase plays important roles in the polyadenylation of viral RNA, the fidelity of RNA replication, ribavirin sensitivity and viral RNA recombination. In other experiments, we identified a group C enterovirus RNA involved in the inhibition of ribonuclease L, an antiviral endoribonuclease. The RNase L ciRNA plays important but largely unexplored roles in pathogenesis. Using novel deep sequencing methods, we found that RNase L targets viral RNA encoding neutralizing epitopes of capsid proteins, sparing most other regions of viral RNA. These data suggest an important interplay between neutralizing antibodies, neutralization escape mutations and antiviral endoribonucleases. A better understanding of viral RNA recombination, enterovirus species groups and antiviral endoribonucleases should help achieve and maintain poliovirus eradication.

HTLV Tax repress nonsense mediated mRNA decay by inhibiting the ATPase activity of UPF1

The Nonsense Mediated mRNA Decay (NMD) is a mRNA quality surveillance pathway that regulates the stability of mRNAs harboring a premature termination codon, avoiding the production of truncated proteins with potential oncogenic properties. NMD regulates also the expression levels of 10 to 15% of human genes such as those regulating the hematopoietic cell differentiation, the maintenance of chromosome structure and function and retroviral RNA. Due to its role in the control of expression of gene involved in the immune response, and the targeting of viral RNA, NMD is now also considered as a viral restriction mechanism. Describing the details of the interplay between HTLV and the NMD is then important to better understand how the virus evades this surveillance and to determine a role of the NMD in the associated pathologies. At the center of the NMD pathway, UPF1 orchestrates a dynamic network of interactions leading to its own structural rearrangement as well as the ribonucleoprotein (RNP) remodeling. In particular, its N-terminal domain (CH domain) repress its ATPase and helicase acitvities held by the central domain (HD domain) until it interacts with the other core factor UPF2 in the early steps of NMD. We showed for the first time that the retrovirus HTLV-1 was able to protect its RNA and modulate some of its transcriptional activators by inhibiting NMD. Now we focuse our attention in deciphering the mechanistics of this inhibition. Thus we demonstrate that HTLV-1 Tax protein targets the RNA helicase UPF1. We found that in cells transfected with a complete provirus, Tax is associated to the NMD sensitive mRNA in an UPF1 dependent manner. This interaction between Tax and the HD domain of UPF1 inhibits the ATPase acivity of UPF1. The homology in amine acid sequence as well as competition assays lead us to the conclusion that Tax could mimics the CH domain in order to maintain a repressive effect even in the latter steps of the pathway. This conclusion is supported in vivo by the accumulation of intermediary complexes stalled on an NMD target. Finally we identified the Tax binding site necessary for this inhibition. Then those data provide structural insights on the regulation of the RNA helicase UPF1 functionning as well as a precise mechanistic of Tax inhibitory effect during NMD.

Mice transgenic for equine cyclin T1 and ELR1 are susceptible to equine infectious anemia virus infection.

BackgroundAs a member of the tumor necrosis factor receptor (TNFR) protein superfamily, equine lentivirus receptor 1 (ELR1) has been shown to be expressed in various equine cells that are permissive for equine infectious anemia virus (EIAV) replication. The EIAV Tat protein (eTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter through a unique mechanism that requires the recruitment of the equine cyclin T1 (eCT1) cofactor into the viral TAR RNA target element. In vitro studies have demonstrated that mouse fibroblast cell lines (e.g., NIH 3T3 cells) that express the EIAV receptor ELR1 and eCT1 support the productive replication of EIAV. Therefore, we constructed transgenic eCT1- and ELR1-expressing mice to examine whether they support in vivo EIAV replication.FindingsFor the first time, we constructed mice transgenic for ELR1 and eCT1. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis confirmed that ELR1 and eCT1 were expressed in the transgenic mouse tissues, particularly in the intestines, spleen and lymph nodes. Consistent with the results of EIAV infection in NIH 3T3 cells expressing ELR1 and eCT1, mouse embryonic fibroblasts (MEFs) from the transgenic mice could support EIAV replication. More importantly, this virus could infect and replicate in mouse blood monocyte-derived macrophages (mMDMs). Macrophages are the principle target cell of EIAV in its natural hosts. Furthermore, after the transgenic mice were inoculated with EIAV, the virus could be detected not only in the plasma of the circulating blood but also in multiple organs, among which, the spleen and lymph nodes were the predominant sites of EIAV replication. Finally, we found that consistent with high viral replication levels, the relevant pathological changes occurred in the spleen and lymph nodes.ConclusionsOur results show that mice transgenic for ELR1 and eCT1 are susceptible to EIAV infection and replication. Further, EIAV infection can cause lesions on the spleen and lymph nodes, similar to those frequently observed in horses, the natural hosts. Therefore, ELR1 and eCT1 are essential in vivo for EIAV invasion and replication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0163-7) contains supplementary material, which is available to authorized users.

Cas9-mediated targeting of viral RNA in eukaryotic cells

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense.

Middle Eastern Respiratory Syndrome Corona Virus (MERS CoV): case reports from a tertiary care hospital in Saudi Arabia.

BACKGROUND AND OBJECTIVESMiddle Eastern respiratory syndrome caused by novel coronavirus (MERS CoV) has been a major public health challenge since it was first described in 2012 in Saudi Arabia. So far, there is no effective treatment for this serious illness, which features a high mortality rate. We report an initial experience of the use of ribavirin and interferon (IFN)-α2b in the management of MERS CoV at a tertiary care hospital.DESIGN AND SETTINGSA case series of 6 patients admitted with a confirmed diagnosis of MERS CoV were treated with ribavirin and IFN-α2b in addition to supportive management. The patients’ demographics, clinical parameters, and outcomes were recorded. Fifty-four close contacts of these patients were screened for MERS CoV.METHODSSix patients with MERS CoV infection were included in this study. Four cases featured symptomatic disease, including pneumonia and respiratory failure, while 2 were asymptomatic close contacts of the MERS CoV patients. The MERS CoV infection was confirmed by reverse transcription–polymerase chain reaction detection of the consensus viral RNA targets upstream of the E gene (UPE) and open reading frame (ORF1b) on a sputum sample. The patients’ demographics, comorbid conditions, time to diagnosis and initiation of treatment, and clinical outcomes were recorded.RESULTSThree out of 6 patients who had comorbid conditions died during the study period, while 3 had successful outcomes. The diagnosis and treatment was delayed by an average of 15 days in those patients who died. Only 2 close contacts out of the 54 screened (3.7%) were positive for MERS CoV.CONCLUSIONTreatment with ribavirin and IFN-α2b may be effective in patients infected with MERS CoV. There appears to be a low infectivity rate among close contacts of MERS CoV patients.

Screening and confirmatory testing strategies for the major transfusion‐transmissible viral infections

BackgroundTesting donated blood for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T‐cell lymphotropic virus (HTLV) is important as all are transfusion‐transmissible viral infections (TTVIs). A particular challenge in donor screening for TTVIs is that donor selection seeks to minimize the prevalence of TTVIs which in the context of testing substantially increases the likelihood that reactive test results represent ‘false’ rather than ‘true’ reactivity. To address this donor testing algorithms seek to combine strategies to optimize both sensitivity (optimal detection of the ‘target’ pathogen) and specificity (minimising ‘false’ reactivity)AimsFirst, review the fundamental principles underpinning TTVI screening and confirmatory algorithms and discuss key challenges. Second, using working examples illustrate how sound design can lead to optimal, cost‐effective solutions.Materials and MethodsScreening algorithms vary dependent on a number of factors including the epidemiology of the TTVI in the donor population and the available resources (e.g. funding, automation level, technical skill). TTVI screening tests include rapid tests (RT), immunoassays (IA) and nucleic acid tests (NAT). RTs and IAs target ‘serological’ markers of infection (anti‐HIV, HIV p24 antigen, anti‐HCV, HBsAg, anti‐HBc or anti‐HTLV) whereas NAT targets viral RNA (HCV and HIV) or DNA (HBV). NAT has the advantage of earlier detection because viral RNA/DNA appears in the bloodstream before serological markers. However NAT is comparatively expensive requiring higher levels of automation and more skilled staff. Confirmatory testing seeks to ‘confirm’ the presence of the pathogen of interest optimizing donor counselling and referral. The appropriate confirmatory strategy will be dependent primarily on the screening strategy and available resourcing.ResultsWhile optimal screening strategies for HIV, HCV and HBV generally include NAT for its superior ability to identify donors with early infection, there are alternative strategies which can provide almost equivalent safety. For example, the use of HIV and HCV ‘combo’ IAs which combine antibody with antigen (HIV p24Ag or HCV Ag) detection significantly improve sensitivity for early infection. In resource poor settings RT for HIV, HCV and to a lesser extent HBV can still provide a high level of safety particularly where carefully selected. Repeating samples reactive on a primary screening test on a second screening test (termed a ‘sequential immunoassay’ or SI algorithm) and assigning a final status based on concordant reactivity has been shown to improve effectiveness although is dependent on judicious test selection. For HIV, HCV and HTLV antibody based screening strategies immunoblots are often used to confirm the presence of antibody. However, immunoblots are relatively costly and can have poor specificity making it important to optimise the specificity of the screening algorithm. Immunoblotting subsequent to a sequential immunoassay approach has been shown to improve the efficiency and effectiveness of the strategy. Since NAT for HIV, HCV and HBV have demonstrated very high specificity, they now form an independent method of confirming infection for serology reactive samples. In some countries, samples concordantly reactive on serology and NAT screening tests are considered ‘confirmed’ without the need for immunoblotting.ConclusionsEven in resource constrained settings optimally designed screening and confirmatory strategies can significantly reduce the risk of transfusion‐transmissible viral infection.

Structure of human RNase L reveals the basis for regulated RNA decay in the IFN response.

One of the hallmark mechanisms activated by type I interferons (IFNs) in human tissues involves cleavage of intracellular RNA by the kinase homology endoribonuclease RNase L. We report 2.8 and 2.1 angstrom crystal structures of human RNase L in complexes with synthetic and natural ligands and a fragment of an RNA substrate. RNase L forms a crossed homodimer stabilized by ankyrin (ANK) and kinase homology (KH) domains, which positions two kinase extension nuclease (KEN) domains for asymmetric RNA recognition. One KEN protomer recognizes an identity nucleotide (U), whereas the other protomer cleaves RNA between nucleotides +1 and +2. The coordinated action of the ANK, KH, and KEN domains thereby provides regulated, sequence-specific cleavage of viral and host RNA targets by RNase L.

Inhibition of influenza virus internalization by (−)-epigallocatechin-3-gallate

(−)-Epigallocatechin-3-gallate (EGCG), one of the major flavonoid components of green tea, is known to have a broad antiviral activity against several enveloped viruses, including the influenza virus. However, its mode of action and the mechanism that allows it to target influenza virus molecules have not been fully elucidated. Thus, this study investigated the molecular mechanism by which EGCG suppresses influenza virus infections. EGCG was found to block an early step in the influenza viral life cycle, but it did not affect viral adsorption to target cells or viral RNA replication. However, EGCG inhibited hemifusion events between virus particles and the cellular membrane by reducing the viral membrane integrity, thereby resulting in the loss of the cell penetration capacity of the influenza virus. EGCG also marginally suppressed the viral and nonviral neuraminidase (NA) activity in an enzyme-based assay system. In conclusion, it is suggested that the anti-influenza viral efficacy of EGCG is attributable to damage to the physical properties of the viral envelope and partial inhibition of the NA surface glycoprotein. These results may facilitate future investigations of the antiviral activity of EGCG against other enveloped viruses as well as influenza virus.