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Emerging antiviral resistant strains of influenza A virus are greatly limiting the therapies available to stop aggressive infections. Genome changes that confer resistance to the two classes of approved antivirals have been identified in circulating influenza A viruses. It is only a matter of time before the currently approved influenza A antivirals are rendered ineffective, emphasizing the need for additional influenza antiviral therapies. This review highlights the current state of antiviral resistance in circulating and highly pathogenic influenza A viruses and explores potential antiviral targets within the proteins of the influenza A virus ribonucleoprotein (vRNP) complex, drawing attention to the viral protein activities and interactions that play an indispensable role in the influenza life cycle. Investigation of small molecule inhibition, accelerated by the use of crystal structures of vRNP proteins, has provided important information about viral protein domains and interactions, and has revealed many promising antiviral drug candidates discussed in this review.
Background: Annually, rubella virus (RV) still causes severe congenital defects in around 100 000 children globally. An attempt to eradicate RV is currently underway and analytical tools to monitor the global decline of the last remaining RV lineages will be useful for assessing the effectiveness of this endeavour. RV evolves rapidly enough that much of this information might be inferable from RV genomic sequence data. Methods: Using BEASTv1.8.0, we analysed publically available RV sequence data to estimate genome-wide and gene-specific nucleotide substitution rates to test whether current estimates of RV substitution rates are representative of the entire RV genome. We specifically accounted for possible confounders of nucleotide substitution rate estimates, such as temporally biased sampling, sporadic recombination, and natural selection favouring either increased or decreased genetic diversity (estimated by the PARRIS and FUBAR methods), at nucleotide sites within the genomic secondary structures (predicted by the NASP method). Results: We determine that RV nucleotide substitution rates range from 1.19 x 10-3 substitutions/site/year in the E1 region to 7.52 x 10-4 substitutions/site/year in the P150 region. We find that differences between substitution rate estimates in different RV genome regions are largely attributable to temporal sampling biases such that datasets containing higher proportions of recently sampled sequences, will tend to have inflated estimates of mean substitution rates. Although there exists little evidence of positive selection or natural genetic recombination in RV, we show that RV genomes possess pervasive biologically functional nucleic acid secondary structure and that purifying selection acting to maintain this structure contributes substantially to variations in estimated nucleotide substitution rates across RV genomes. Conclusion: Both temporal sampling biases and purifying selection favouring the conservation of RV nucleic acid secondary structures have an appreciable impact on substitution rate estimates but do not preclude the use of RV sequence data to date ancestral sequences. The combination of uniformly high substitution rates across the RV genome and strong temporal structure within the available sequence data, suggests that such data should be suitable for tracking the demographic, epidemiological and movement dynamics of this virus during eradication attempts.
Background: The antiviral therapy of chronic hepatitis B virus (HBV) infection pursues the dual goals, virological response (undetectable serum HBV DNA) and hepatitis B e antigen (HBeAg) serological response (serum HBeAg loss/seroconversion). It is relatively difficult, however, to realize the serological response, especially for nucleotide/nucleoside analogs. Furin, a proprotein convertase, is involved in HBeAg maturation. The suppression of furin using inhibitors accordingly reduces HBeAg secretion, but possibly enhances HBV replication. For these reasons, the strategy based on the combination of nucleoside analog entecavir (ETV) and furin inhibitors to inhibit HBV replication and HBeAg secretion simultaneously were studied here. Methods: The suppression of furin was performed using inhibitors decanoyl-RVKR-chloromethylketone (CMK) and hexa-D-arginine (D6R) or the expression of furin inhibitory prosegment. The influence of furin suppression on HBV replication and the effect of CMK combined with nucleoside analog entecavir (ETV) on HBV replication and HBeAg secretion was investigated in HepG2.2.15 cells. HBeAg level in media was detected using enzyme-linked immunosorbent assay. Intracellular viral antigens and HBV DNA were detected using Western and Southern blotting analyses, respectively. Results: CMK, D6R and the expression of inhibitory prosegment all significantly reduced HBeAg secretion, but only CMK enhance HBV replication. Concordantly, only CMK post-transcriptionally accumulated cytosolic HBV replication-essential hepatitis B core antigen (HBcAg). The HBcAg-accumulating effect of CMK was further found to be resulted from its redundant inhibitory effect on the trypsin-like activity of cellular proteasomes that are responsible for HBcAg degradation. Moreover, the viral replication-enhancing effect of CMK was abrogated by ETV and ETV combined with CMK reduced HBV replication and HBeAg secretion simultaneously. Conclusion: The suppression of furin itself does not enhance HBV replication. Nucleotide/nucleoside analogs combined with furin inhibitors may be a potential easy way to realize the dual goals of the antiviral therapy for chronic hepatitis B in the future.
Background: Dengue is the most frequent arthropod-borne viral disease worldwide. Because dengue manifestations are similar to those of many other febrile syndromes, the availability of dengue-specific laboratory tests is useful for the differential diagnosis. Timely and accurate diagnosis of dengue virus (DENV) infection is important for appropriate management of complications, pathophysiological studies, epidemiological investigations and optimization of vector-control measures. Several qquot;in-houseqquot; reverse transcriptase-polymerase chain reaction (RT-PCR) methods have been developed to detect, type and/or quantify DENV. Standardized dengue RT-PCR kits with internal controls have been recently introduced, but need clinical evaluation. We assessed the performances of 4 commercial DENV real-time RT-PCR kits.FindingsThe 4 kits were evaluated using a panel of 162 samples positive with an existing in-place hemi-nested RT-PCR used for routine DENV-infection diagnosis in patients with acute-febrile disease. The panel included 46 DENV-1, 37 DENV-2, 33 DENV-3, and 46 DENV-4. Also, 70 negative serum specimens were used to determine specificity. Geno-Senaapos;s Dengue 1-4 Real-Time RT-PCR kit was the only assay to provide quantification using standards, but lacked sensitivity for DENV-4 detection. The SimplexaTM Dengue RT-PCR assay, with 151 (93.2% [95% confidence interval, 89.3-97.1]) positive samples, had significantly higher sensitivity than the other 3 kits; in a complementary evaluation of 111 consecutive patientsaapos; samples, its performance and genotyping agreed with the hemi-nested gold-standard assay. Conclusions: The SimplexaTM Dengue RT-PCRaapos;s good performance to detect and genotype DENV1-4 requires further evaluation in multicenter and prospective studies, particularly in settings of clinical diagnosis during dengue outbreaks.
Background: Major histocompatibility complex (MHC) class I peptide binding and presentation are essential for antigen-specific activation of cytotoxic T lymphocytes (CTLs) and swine MHC class I molecules, also termed swine leukocyte antigens (SLA), thus play a crucial role in the process that leads to elimination of viruses such as swine influenza virus (SwIV). This study describes the identification of SLA-presented peptide epitopes that are targets for a swine CTL response, and further analyses multiple specificities expressed by SwIV activated CTL subsets.FindingsFour SwIV derived peptides were identified as T cell epitopes using fluorescent influenza:SLA tetramers. In addition, multiple CTL specificities were analyzed using peptide sequence substitutions in two of the four epitope candidates analyzed. Interestingly both conserved and substituted peptides were found to stain the CD4-CD8+ T cell subsets indicating multiple specificities. Conclusions: This study describes a timely and cost-effective approach for viral epitope identification in livestock animals. Analysis of T cell subsets showed multiple specificities suggesting SLA-bound epitope recognition of different conformations.
Background: Hepatitis E is a disease of major public-health concern mainly in developing countries. Although molecular and sero-epidemiological investigations of HEV have been performed in many provinces in China, the epidemiological data from Yunnan Province are limited and genotypes are not be fully characterized. In this study the prevalence and characteristics of hepatitis E virus (HEV) detected in pigs from Yunnan province, China was evaluated. Results: A total of 13 out of 187 pig fecal samples collected in 2011 revealed HEV positive results; likewise, 7 out of 69 samples collected in 2012 exhibited positive results. These findings indicated a total prevalence of 7.8% (20/256). Phylogenetic and molecular evolutionary analysis results revealed that nine strains were found in the samples obtained in 2011, in which 87.1% to 99.4% nucleotide sequence identity was shared among these strains; and 77.0% to 81.9%, 52.2% to 53.6%, 77.0% to 88.2% and 77.9% to 96.8% nucleotide sequence identities were shared with strains representing genotypes 1, 2, 3, and 4. Five strains were detected in the samples obtained in 2012, in which 94.2% to 99.3% nucleotide sequence identity was shared among the strains, and 81.0% to 82.5%, 81.8% to 83.2%, 81.0% to 92.7% and 81.0% to 97.8% nucleotide sequence identities were shared with strains representing the genotypes 1, 2, 3, and 4. Conclusions: Analysis of fourteen detected HEV strains revealed that three of them were subtype 4d, two were subtype 4b; the nine remaining isolated strains were subtype 4 h. These results indicated that the prevalence of HEV in the swine herds of Yunnan was quite high, additional public-health concerns should focus on pork safety.
Background: Borna disease virus (BDV) is an evolutionary old RNA virus, which infects brain and blood cells of humans, their primate ancestors, and other mammals. Human infection has been correlated to mood disorders and schizophrenia, but the impact of BDV on mental-health still remains controversial due to poor methodological and cross-national comparability.MethodThis first report from the Middle East aimed to determine BDV infection prevalence in Iranian acute psychiatric disorder patients and healthy controls through circulating immune complexes (CIC), antibodies (Ab) and antigen (pAg) in blood plasma using a standardized triple enzyme immune assay (EIA). Samples of 314 subjects (114 psychiatric cases, 69 blood donors, and 131 healthy controls) were assayed and data analyzed quantitatively and qualitatively. Results: CICs revealed a BDV prevalence of one third (29.5%) in healthy Iranian controls (27.5% controls; 33.3% blood donors). In psychiatric patients CIC prevalence was higher than in controls (40.4%) and significantly correlating with bipolar patients exhibiting overt clinical symptoms (p = 0.005, OR = 1.65). CIC values were significantly elevated in bipolar (p = 0.001) and major depressive disorder (p = 0.029) patients as compared to controls, and in females compared to males (p = 0.031). Conclusion: This study supports a similarly high prevalence of subclinical human BDV infections in Iran as reported for central Europe, and provides again an indication for the correlation of BDV infection and mood disorders. Further studies should address the morbidity risk for healthy carriers and those with elevated CIC levels, along with gender disparities.
Background: The signaling pathways most critical to prion disease pathogenesis are as yet incompletely characterized. We have developed a kinomics approach to identify signaling pathways that are dysregulated during prion pathogenesis. The approach is sensitive and specific enough to detect signaling pathways dysregulated in a simple in vitro model of prion pathogenesis. Here, we used this approach to identify signaling pathways dysregulated during prion pathogenesis in vivo. Methods: Mice intraperitoneally infected with scrapie (strain RML) were euthanized at 70, 90, 110, 130 days post-infection (dpi) or at terminal stages of disease (155–190 dpi). The levels of 139 protein kinases in brainstem-cerebellum homogenates were analyzed by multiplex Western blots, followed by hierarchical clustering and analyses of activation states. Results: Hierarchical and functional clustering identified CaMK4β and MST1 signaling pathways as potentially dysregulated. Targeted analyses revealed that CaMK4β and its downstream substrate CREB, which promotes neuronal survival, were activated at 70 and 90 dpi in cortical, subcortical and brainstem-cerebellum homogenates from scrapie-infected mice. The activation levels of CaMK4β/CREB signaling returned to those in mock-infected mice at 110 dpi, whereas MST1, which promotes neuronal death, became activated at 130 dpi. Conclusion: Pro-survival CaMK4β/CREB signaling is activated in mouse scrapie at earlier times and later inhibited, whereas pro-death MST1 signaling is activated at these later times.
Background: H3N8 equine influenza virus (EIV) has caused disease outbreaks in horses across the world since its first isolation in 1963. However, unlike human, swine and avian influenza, there is relatively little sequence data available for this virus. The majority of published sequences are for the segment encoding haemagglutinin (HA), one of the two surface glycoproteins, making it difficult to study the evolution of the other gene segments and determine the level of reassortment occurring between sub-lineages. Methods: To facilitate the generation of full genome sequences for EIV, we developed a simple, cost-effective and efficient method. M13-tagged primers were used to amplify short, overlapping RT-PCR products, which were then sequenced using Sanger dideoxynucleotide sequencing technology. We also modified a previously published method, developed for human H3N2 and avian H5N1 influenza viruses, which was based on the ligation of viral RNA and subsequent amplification by RT-PCR, to sequence the non-coding termini (NCRs). This necessitated the design of novel primers for an N8 neuraminidase segment. Results: Two field isolates were sequenced successfully, A/equine/Lincolnshire/1/07 and A/equine/Richmond/1/07, representative of the Florida sublineage clades 1 and 2 respectively. A total of 26 PCR products varying in length from 400–600 nucleotides allowed full coverage of the coding sequences of the eight segments, with sufficient overlap to allow sequence assembly with no primer-derived sequences. Sequences were also determined for the non-coding regions and revealed cytosine at nucleotide 4 in the polymerase segments. Analysis of EIV genomes sequenced using these methods revealed a novel polymorphism in the PA-X protein in some isolates. Conclusions: These methods can be used to determine the genome sequences of EIV, including the NCRs, from both clade 1 and clade 2 of the Florida sublineage. Full genomes were covered efficiently using fewer PCR products than previously reported methods for influenza A viruses, the techniques used are affordable and the equipment required is available in most research laboratories. The adoption of these methods will hopefully allow for an increase in the number of full genomes available for EIV, leading to improved surveillance and a better understanding of EIV evolution.
Background: JC polyomavirus (JCPyV) is a widespread human polyomavirus that usually resides latently in its host, but can be reactivated under immune-compromised conditions potentially causing Progressive Multifocal Leukoencephalopathy (PML). JCPyV encodes its own microRNA, jcv-miR-J1. Methods: We have investigated in 50 healthy subjects whether jcv-miR-J1-5p (and its variant jcv-miR-J1a-5p) can be detected in plasma or urine. Results: We found that the overall detection rate of JCPyV miRNA was 74% (37/50) in plasma and 62% (31/50) in urine. Subjects were further categorized based on JCPyV VP1 serology status and viral shedding. In seronegative subjects, JCPyV miRNA was found in 86% (12/14) and 57% (8/14) of plasma and urine samples, respectively. In seropositive subjects, the detection rate was 69% (25/36) and 64% (23/36) for plasma and urine, respectively. Furthermore, in seropositive subjects shedding virus in urine, higher levels of urinary viral miRNAs were observed, compared to non-shedding seropositive subjects (P llt; 0.001). No correlation was observed between urinary and plasma miRNAs. Conclusion: These data indicate that analysis of circulating viral miRNAs divulge the presence of latent JCPyV infection allowing further stratification of seropositive individuals. Also, our data indicate higher infection rates than would be expected from serology alone.