Capacity building permitting comprehensive monitoring of a severe case of Lassa hemorrhagic fever in Sierra Leone with a positive outcome. Part 2
Human Subjects: Suspected LF patients, close contacts, and healthy volunteers were eligible to participate in these studies as outlined in Tulane University’s Institutional Review Board (IRB) protocol for this project, National Institutes of Health/National Institutes of Allergy and Infectious Diseases guidelines governing the use of human subject for research, and Department of Health and Human Services/National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Numbers AI067188 and AI082119. This project was approved by the Tulane University IRB. Adult patients in this manuscript have given written informed consent for the publication of their case details. Written informed consent was obtained from the adult guardian of patient G-1180 for publication of this case report.
Normal and positive control sera: Serum from one Sierra Leonean and two Caucasian American volunteers were used in these studies as normal controls. A serum sample collected from a 20-year-old pregnant woman who succumbed to Lassa fever in the KGH Maternity Ward on August 29, 2010 was used as a positive control. A single serum sample was collected from this subject before expiration, and assigned the coded designation G-1177.
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Detection of Lassa virus antigen and Lassa virus-specific antibodies: Serum levels of Lassa nucleoprotein (NP)-specific antigen were first determined using LFI modules currently under pre-clinical development by Corgenix Medical Corp., Broomfield, CO, U.S.A., and the Lassa fever consortium (see acknowledgements). The LFI modules utilize LASV NP specific murine monoclonal antibodies in a capture line and gold-conjugated detection reagent. Downstream of the capture line is an anti-murine polyclonal control line. Serum samples are added to a sample well followed by buffer solution to initiate lateral flow through the reagent pads and across the capture and control lines. The formation of Lassa NP antigen immune-complexes by the reagents produces a red signal due to gold conjugate deposition, allowing visual interpretation or measurement by reflectance. The red signal can be seen within 3-5 minutes with full signal development between 15-25 minutes. The positive Lassa fever diagnosis is then confirmed with a sensitive antigen-capture ELISA employing either a murine monoclonal or caprine polyclonal capture antibody, followed by a peroxidase-labeled caprine reagent and tetramethylbenzidine (TMB) substrate. A standard curve was generated with recombinant LASV NP in order to quantify serum levels of virus-associated NP.
IgM and IgG levels to recombinant LASV proteins (NP alone and NP – glycoprotein 1 (GP1) – glycoprotein complex (GPC) combination) ELISA: Individual LASV proteins and combinations optimized for detection of virus-specific IgM and IgG levels in serum were coated in stripwell plates, blocked, dried, and packaged with desiccating packs (Corgenix Medical Corp.). For analysis, sera were diluted 1:100 in a proprietary sample dilution buffer and incubated in wells for 30 minutes at room temperature, washed, and incubated with optimized HRP-labeled anti-human IgG or IgM conjugates for an additional 30 minutes. After washing, detection was performed with TMB substrate for 10 minutes at room temperature, stopped with sulphuric acid, and read at A450 in a BioTek ELISA plate reader.
Capacity building permitting comprehensive monitoring of a severe case of Lassa hemorrhagic fever in Sierra Leone with a positive outcome
Abstract
Lassa fever is a neglected tropical disease with a significant impact on the health care system of endemic West African nations. To date, case reports of Lassa fever have focused on laboratory characterisation of serological, biochemical and molecular aspects of the disease imported by infected individuals from Western Africa to the United States, Canada, Europe, Japan and Israel. Our report presents the first comprehensive real time diagnosis and characterization of a severe, hemorrhagic Lassa fever case in a Sierra Leonean individual admitted to the Kenema Government Hospital Lassa Fever Ward. Fever, malaise, unresponsiveness to anti-malarial and antibiotic drugs, followed by worsening symptoms and onset of haemorrhaging prompted medical officials to suspect Lassa fever. A recombinant Lassa virus protein based diagnostic was employed in diagnosing Lassa fever upon admission. This patient experienced a severe case of Lassa hemorrhagic fever with dysregulation of overall homeostasis, significant liver and renal system involvement, the interplay of pro- and anti-inflammatory cytokines during the course of hospitalization and an eventual successful outcome. These studies provide new insights into the pathophysiology and management of this viral illness and outline the improved infrastructure, research and real-time diagnostic capabilities within LASV endemic areas.
Background
Lassa virus (LASV), a member of the Arenaviridae family, is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. There are an estimated 300,000 – 500,000 cases of Lassa fever each year with a mortality rate of 15%-20% for hospitalized patients, which can become as high as 50% during epidemics. Presently, there is no licensed vaccine or immunotherapy available for prevention or treatment of this disease. Although the antiviral drug ribavirin is somewhat beneficial, it must be administered at an early stage of infection to successfully alter disease outcome, thereby limiting its utility. Furthermore, there is no commercially available Lassa fever diagnostic assay, which hampers early detection and rapid implementation of existing treatment regimens (e.g. ribavirin administration). The severity of the disease, its ability to be transmitted by aerosol droplets and the lack of a vaccine or therapeutic drug led to its classification as a National Institutes of Allergy and Infectious Diseases (NIAID) Category A pathogen and biosafety level-4 (BSL-4) agent. Several imported Lassa fever cases have been described since 1973 primarily from foreign nationals displaying signs of the disease upon returning to native countries or having been evacuated after falling ill abroad. To date, and despite the often severe nature of Lassa fever in Western African nations, resources have not been available to perform comprehensive daily analysis of blood samples from suspected and confirmed patients in-country. Continuous infrastructure improvements at the Kenema Government Hospital (KGH) Lassa Fever Laboratory (LFL) by Tulane University, Department of Defense (DoD) and the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 2005 have allowed for the implementation of sophisticated diagnostic and research capabilities at this location. Currently the KGH LFL diagnoses Lassa fever using ELISA and lateral flow immunoassay (LFI) platforms to detect viral antigen and virus-specific IgM and IgG levels in the serum of every suspected case presented to the LFW. Additionally, the laboratory can assess 14 serum analytes using a Piccolo® blood chemistry analyzer coupled with comprehensive metabolic panel disks. Flow cytometry powered by a 4-color Accuri® C6 cytometer is used to perform immunophenotyping and intracellular and bead-based secreted cytokine analysis. Together, these diagnostic assays and instruments enabled the analysis of metabolic and inflammatory functions in real time utilizing the sera of individuals discussed in this case report with concomitant appropriate medical intervention.
The main patient case discussed in this report was closely monitored for nine days during his hospitalization, during which time his condition stabilized; he began walking with supervision and was nearing the end of ribavirin treatment. These studies herald a new era in real time diagnosis and management of Lassa hemorrhagic fever in resource poor, endemic areas of Western Africa. They represent a novel platform toward more efficient and broader control of the effects of this disease in the population at large.
Comparative genomics of the Microviridae. Part 4
The ΦCA82 genome and ORFs were aligned with selected microvirus sequences using ClustalW [26]. Putative ORFs within the ΦCA82 genome were predicted using the FGENESV Trained Pattern/Markov chain-based viral gene prediction method from the Softberry website. Searches for conserved domains within the ΦCA82 genome were performed with the Conserved Domain Database (CDD) Search Service v2.17 at the National Center for Biotechnology Information (NCBI) website.
Comparative genomics of the Microviridae
The sequence of phage ΦCA82 was compared to 14 other members of the Microviridae (Table 1) obtained from the integrated microbial genomes (IMG) system [29]. To first determine nucleotide level similarities, tetra-nucleotide comparisons between genomes were performed with jspecies [30]. Pairwise genome comparisons were based on regressions of normalized tetra-nucleotide frequency counts and the distributions of the R2 values from these comparisons were visualized in R. To compare genomes based on similarity of predicted gene sequences, the program CD-HIT [32] was used.
Genomic functional comparisons were based on pfam categories for each predicted gene as classified by the IMG annotation pipeline. A data table of pfam categories and gene counts for each genome was used to construct a similarity matrix and dendrogram in R. To determine which predicted genes were unique to ΦCA82 and those which were shared with other Microviridae members, the Microviridae pangenome was constructed as the union of all predicted genes from the 14 Microviridae genomes and compared to predicted genes for ΦCA82 using both CD-HIT and our data analysis pipeline as described above and blastp run with default parameters except for an e-value cutoff of 0.01.
Results and Discussion
The entire circular, single-stranded nucleotide sequence for the uncultured microvirus ΦCA82 genome was determined to be 5,514 nucleotides. The complete genome sequence had a nucleotide composition of A (38.6%), C (19.6%), G (20.1%), and T (21.6%) with an overall G + C content of 39.7%, which is similar to the chlamydial phages (37-40%). The ΦCA82 genome was organized in a modular arrangement similar to microviruses and encoded predicted proteins homologous to those chlamydial bacteriophage types and to the Bdellovibrio bacteriovorus ΦMH2K. The coding capacity of the genome is 91% as it encodes ten ORFs, greater than 99 nucleotides similarly to other chlamydial microvirus genomes [35]. The genome size, number of ORFs and total coding % of nucleotides as depicted in Figure 1 is larger than most of the chlamydial phages and is closer in size to the ΦX174 genome.
Comparative genomics of the Microviridae. Part 3
Introduction
Metagenomics analyses have lead to the discovery of a variety of microbial nucleotide sequences from environmental samples. These techniques have also allowed for the discovery of uncultured viral nucleotide sequences that are commonly from bacteriophages that has also resulted in the discovery of useful enzymes for molecular biology. There has been a resurgent interest in bacteriophage biology and their use or use of phage gene products as antibacterial agents. Bacteriophages are thought to be the most abundant life form as a group and the importance of phage to bacterial evolution, the role of phage or prophage encoded virulence factors that contribute to bacterial infectious diseases and their contribution to horizontal gene transfer cannot be over stated. Additionally, the contribution to microbial ecology and to agricultural production is also extremely important.
Enteric diseases are an important economic production problem for the poultry industry worldwide. One of the major economically important enteric diseases for the poultry industry are the poult enteritis complex (PEC) and poult enteritis mortality syndrome (PEMS) in turkeys and a runting-stunting syndrome (RSS) in broiler chickens. Consequently, studies have been ongoing to identify novel enteric viruses among poultry species at our laboratory. In a recent study, we utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric disease. This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys.
Additionally, we have successfully applied a random PCR-based method for detection of unknown microorganisms from enteric samples of turkeys that resulted in identification of genomic sequences and subsequent determination of the full-length genome from a previously uncultured parvovirus. During these ongoing investigations to further characterize the turkey gut microbiome and identify novel viral pathogens of poultry, bacteriophage genomic sequences have also been identified. Herein we report the complete genomic sequence of a putative novel member of the Microviridae obtained from turkey gastrointestinal DNA samples utilizing metagenomics approaches. The protein sequences of ΦCA82 were most similar to those of Chlamydia phages.
Forty-two complete intestinal tracts (from duodenum/pancreas to cloaca, including cecal tonsils) from a turkey farm in California, U.S.A. with histories of enteric disease problems were received at the Southeast Poultry Research Laboratory (SEPRL). The intestines were processed and pooled into a single sample, as previously described. A sequence-independent polymerase chain reaction (PCR) protocol was employed to amplify particle-associated nucleic acid (PAN) present in turkey intestinal homogenates, and has been described elsewhere in detail. Using this approach, a total of 576 clones were identified and sequenced with the M13 forward and reverse primers on an AB-3730 automated DNA sequencer. The sequenced clones were used as query sequences to search the GenBank non-redundant nucleotide and protein databases using the blastn and blastx algorithms. In total, the majority of clones with inserts had no hit in the databases using tblastx. However, 46% of the cloned DNA had homology to cellular DNA, bacterial DNA, bacteriophage DNA, and several eukaryotic viral DNA genomes. Twelve DNA clones had sequence similarity to single-stranded DNA microphages, which have also been identified predominantly in microbialites. A contig, CA82 with an average of eightfold coverage and length of 1962 nt was assembled from eight of those clones. This contig had no significant nucleotide similarity to database sequences, but the deduced amino acid sequence revealed significant similarity to the members of the family Microviridae. This initial contig was used to design PCR primers in the opposite orientation of the circular ssDNA to assemble into a contiguous ΦCA82 genome. The PCR amplification resulted in a 3.4 kb product that closed the gap between the CA82 contig and the rest of the circular genome. The final sequence was confirmed by sub-cloning and primer walking with primers resulting ~1 kb fragments containing 250 bp overlapping sequences across the genome. The circular DNA genome was assembled from contigs exhibiting 100% nucleotide identity within the overlapping regions.
Comparative genomics of the Microviridae. Part 2
Chipman et al predicted that the IN5 trimer structure in VP1 may function as a substitute for spike proteins of the ΦX174-like viruses, which are not found in SpV4 or the Chlamydia phages, and as such may be responsible for receptor recognition. It has also been suggested that the diverse sequence in this region is associated with host range of phages. The presence of a large insertion in ΦCA82 further supports that it is closer to the intracellular phage subfamily and the sequence dissimilarity within this region between the ΦCA82 and various other phages strongly indicates that this domain indeed may function as a host range determinant.
Rep protein of ΦCA82
ORF3 encoded a putative replication initiation protein that was most similar to the SpV4-rep and the Bdellovibrio phage ΦMH2K-rep [36] proteins. Pairwise alignment of the ΦCA82 VP3 (rep) protein and SpV4 p1 (rep) protein revealed the presence of two conserved domains from residues 73 through 176 and 195 through 320 of the ΦCA82 protein. Overall, the two rep proteins only had 22.6% identity, but shared many of the same sequences throughout the conserved regions that were recognized by BLAST as putative replication initiation protein regions. Rep protein plays an essential role in viral DNA replication and binds the origin of replication where it cleaves the dsDNA replicative form I (RFI) and becomes covalently bound to it via phosphotyrosine bond. The conservation of the functional domains between the ΦCA82 phage rep protein and other microviral replication initiation proteins suggests a similar pathway/mechanism for DNA replication and virion packaging.
Full genome comparisons of ΦCA82 with other members of the Microviridae
The ΦCA82 genome is quite different from other members of Microviridae as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Comparisons of ΦCA82 to 14 other Microviridae genomes showed very low correlations of tetra-nucleotide frequencies as a measure of genome similarity. ΦCA82 was most similar to SpV4, but the correlation of tetra-nucleotide frequencies was poor (R2 = 0.33; Figure 4A). Only ΦMH2K had lower similarities to other Microviridae (Figure 4A). Clustering of predicted proteins showed ΦCA82 was most closely related to a clade comprised of the chlamydial phages, but as in the nucleotide comparisons, the predicted proteins of ΦCA82 are quite distinct from those of the other microviruses. Function-based clustering of genomes using pfam categories showed that ΦCA82 was most similar to SpV4, based on shared membership of the ΦCA82 ORF1 in pfam02305, an F super family capsid protein. These results were confirmed by comparisons of predicted proteins from the ΦCA82 genome to a Microviridae pangenome. This analysis showed only three genes with significant similarity as determined by local alignments using blastp with no overlap between ΦCA82 and the Microviridae pangenome based on global alignments at a 40% similarity cutoff. ΦCA82 is only distantly related to other Microviridae, but is most similar to SpV4 and the chlamydial phages. In summary, the whole genome comparisons of ΦCA82 to other Microviridae members indicate a distant evolutionary relationship, perhaps suggesting that the divergence of ΦCA82 from other microviruses reflects unique evolutionary pressures encountered within the turkey gastrointestinal system.
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Comparative genomics of the Microviridae
The sequence of phage ΦCA82 was compared to 14 other members of the Microviridae obtained from the integrated microbial genomes (IMG) system. To first determine nucleotide level similarities, tetra-nucleotide comparisons between genomes were performed with jspecies. Pairwise genome comparisons were based on regressions of normalized tetra-nucleotide frequency counts and the distributions of the R2 values from these comparisons were visualized in R. To compare genomes based on similarity of predicted gene sequences, the program CD-HIT was used.
The entire circular, single-stranded nucleotide sequence for the uncultured microvirus ΦCA82 genome was determined to be 5,514 nucleotides. The complete genome sequence had a nucleotide composition of A (38.6%), C (19.6%), G (20.1%), and T (21.6%) with an overall G + C content of 39.7%, which is similar to the chlamydial phages (37-40%). The ΦCA82 genome was organized in a modular arrangement similar to microviruses and encoded predicted proteins homologous to those chlamydial bacteriophage types and to the Bdellovibrio bacteriovorus ΦMH2K. The coding capacity of the genome is 91% as it encodes ten ORFs, greater than 99 nucleotides similarly to other chlamydial microvirus genomes. The genome size, number of ORFs and total coding % of nucleotides as depicted in Figure 1 is larger than most of the chlamydial phages and is closer in size to the ΦX174 genome.
A total of ten genes could be identified of which only three gene products could be assigned with a known function based upon BLAST analysis. The predicted major capsid protein VP1 encoded by ORF1 belongs to the family of single-stranded bacteriophages and is the major structural component of the virion that may contain as many as 60 copies of the protein. The closest sequence similarity of the 565 amino acid ΦCA82 VP1 protein was with the Spiroplasma phage 4 (SpV4) capsid protein and the chlamydial phage VP1 proteins, as well as the Chlamydia prophage CPAR39 and Bdellovibrio phage ΦMH2K major capsid protein. A putative minor capsid protein of 234 amino acids was encoded by ORF2 that had similarity to the chlamydial bacteriophages and the Bdellovibrio phage ΦMH2K that was originally postulated to be an attachment protein.
Recent studies using a comparative metagenomic analysis of viral communities associated with marine and freshwater microbialites indicated that identifiable sequences in these were dominated by single-stranded DNA microphages [25]. Partial sequence analysis of the VP1 gene from these microphages showed that the similarity between metagenomic clones and cultured microphage capsid sequences ranged from 47.5 to 61.2% at the nucleic-acid level and from 37.2 to 69.3% at the protein level, respectively. Interestingly, the VP1 gene of ΦCA82 has a similarly high level of sequence similarity (69.1% at the amino acid level) with the seawater metagenomic phages within the same VP1 region (data not shown). This observation is consistent with an environmental origin of modern poultry phages that have since undergone significant host-specific evolutionary divergence in agricultural settings.
A multiple alignment of major capsid proteins among diverse members shows similarities within the entire predicted coding region with the exception of the predicted surface-exposed IN5 loop and Ins. Amino acids located within these regions are involved in forming large protrusions at the threefold icosahedral axes of symmetry in the intracellular microvirus phages. The IN5 loop, forming a globular protrusion on the virus coat and is the most variable region in the VP1 proteins from Chlamydia and Spiroplasma phages is potentially located from residues 198 through 295 of ΦCA82 VP1, which is the most highly variable portion of the protein by BLAST. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection. The short variable Ins sequences of ΦCA82 are putatively located from residues 459 through 464 of the VP1 protein.
The complete genome sequence
The genomic DNA sequence of a novel enteric uncultured microphage, ΦCA82 from a turkey gastrointestinal system was determined utilizing metagenomics techniques. The entire circular, single-stranded nucleotide sequence of the genome was 5,514 nucleotides. The ΦCA82 genome is quite different from other microviruses as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Only three genes showed significant similarity to microviral proteins as determined by local alignments using BLAST analysis. ORF1 encoded a predicted phage F capsid protein that was phylogenetically most similar to the Microviridae ΦMH2K member’s major coat protein. The ΦCA82 genome also encoded a predicted minor capsid protein (ORF2) and putative replication initiation protein (ORF3) most similar to the microviral bacteriophage SpV4. The distant evolutionary relationship of ΦCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system.
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Metagenomics analyses have lead to the discovery of a variety of microbial nucleotide sequences from environmental samples. These techniques have also allowed for the discovery of uncultured viral nucleotide sequences that are commonly from bacteriophages that has also resulted in the discovery of useful enzymes for molecular biology. There has been a resurgent interest in bacteriophage biology and their use or use of phage gene products as antibacterial agents. Bacteriophages are thought to be the most abundant life form as a group and the importance of phage to bacterial evolution, the role of phage or prophage encoded virulence factors that contribute to bacterial infectious diseases and their contribution to horizontal gene transfer cannot be over stated. Additionally, the contribution to microbial ecology and to agricultural production is also extremely important.
Enteric diseases are an important economic production problem for the poultry industry worldwide. One of the major economically important enteric diseases for the poultry industry are the poult enteritis complex (PEC) and poult enteritis mortality syndrome (PEMS) in turkeys and a runting-stunting syndrome (RSS) in broiler chickens. Consequently, studies have been ongoing to identify novel enteric viruses among poultry species at our laboratory. In a recent study, we utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric disease. This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys.
Additionally, we have successfully applied a random PCR-based method for detection of unknown microorganisms from enteric samples of turkeys that resulted in identification of genomic sequences and subsequent determination of the full-length genome from a previously uncultured parvovirus. During these ongoing investigations to further characterize the turkey gut microbiome and identify novel viral pathogens of poultry, bacteriophage genomic sequences have also been identified. Herein we report the complete genomic sequence of a putative novel member of the Microviridae obtained from turkey gastrointestinal DNA samples utilizing metagenomics approaches. The protein sequences of ΦCA82 were most similar to those of Chlamydia phages.
Specific inhibition of NP expression by siS1 and siS2. Discussion
Discussion
With the noticeable exception of ribavirin recommended by the World Health Organization (WHO) to treat CCHFV infection, there is no specific medical therapy. Therefore, there is a need for the development of novel antiviral strategies against nairovirus infections.
In this work, for the first time, we evaluated the antiviral activity of siRNAs targeting the L (polymerase), M (glycoproteins) and S (nucleoprotein) transcripts of HAZV, a non pathogenic nairovirus in humans, which is considered as a surrogate CCHFV model.
We observed that siRNAs complementary to the mRNAs encoded by the L and M genomic segments had a lower effect than those targeting the S segment. Interestingly, three studies performed in arthropod cells showed that the nucleoprotein gene of orthobunyaviruses is a RNAi prim target. We demonstrated the efficient inhibition of HAZV in tick cells via RNAi induced by a Semliki Forest replicon expressing the S segment whereas the L segment had no effect. In earlier studies, Billecocq et al. and Powers et al. observed a similar phenomenom in mosquito cells infected by Rift Valley Fever virus and La Crosse virus, respectively. Furthermore, in mammalian cells La Crosse virus replication was successfully decreased by siRNAs targeting the S segment and similarly to our results, the L and M siRNAs had a much weaker effect. Finally, orthobunyavirus Akabane replication was inhibited up to 99% by siRNAs directed against highly conserved regions of the nucleoprotein gene.
As other RNA viruses, nairovirus present a high mutation rate which might contribute to their escape from siRNAs inhibition. The emergence of viral mutants is lowered when using siRNAs targeting the most conserved viral sequences. Among the three genomic segments, the S segment is the most conserved within the Nairovirus genus because of its lowest mutation rate. This observation indicates that siRNAs directed against the S segment are potentially interesting anti-nairovirus molecules.
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The nucleoprotein plays a crucial role in the regulation of viral transcription and replication. It associates with genomic RNA and serves as a template for the polymerase to initiate both steps during virus cycle. It is assumed that cytoplasmic NP concentration is important for the transition from transcription to replication and therefore, a decrease in NP production should contribute to virus inhibition, as observed in our study.
Among all tested siRNAs, only three (siS1, siS2 and siS3) exhibited a strong inhibition of HAZV replication (up to 90%). The unequal siRNAs efficiency is in agreement with previous works. The interaction of proteins with the viral transcripts or the presence of secondary structures in these mRNAs may interfere the proper recognition by the homologous siRNAs, explaining the variability of siRNAs efficacy. Interestingly, the combination of siS1, siS2 and siS3 used at a concentration of 33 nM (100 nM siRNA final concentration) induced ~ 90% virus inhibition which is similar to the effect observed for each individual siRNAs. The absence of additive or synergistic antiviral effect observed here was also reported in other studies. It was hypothesized that the competition of siRNAs for RISC might explain this lack of effect.
In our study, the two most active siRNAs, siS1 and siS2, demonstrated a specific inhibitory effect against HAZV in a dose-dependent manner. These two siRNAs did not induce IFN production or cell toxicity. We showed that treatment with each siRNAs correlates with the reduction of nucleoprotein expression level. A concentration as low as 0.1 nM for both siRNAs was sufficient to reduce significantly HAZV replication. A similar antiviral effect at such concentrations had already been described in a previous study with Epstein-Barr virus. We observed that the siRNA stability lasted at least up to 72 hrs. The prolonged stability of siRNAs with a relatively long half life (from 3 to 8 days after transfection) was also shown in experiments with vaccinia virus, monkeypox virus, food and mouth disease virus and HIV-1. We also demonstrated that the antiviral activity of siRNAs was effective when carried out after infection. Taken together, the results are encouraging for the use of siRNA as future prophylactic and therapeutic treatments of nairovirus infection.
Inhibition of Hazara nairovirus replication by small interfering RNAs. Part 2
The nairovirus are spherical enveloped particles of 100 nm in diameter. Their genome consists of three segments of single-stranded RNA of negative polarity designated S (Small), M (Medium) and L (Large) RNA segments. These three segments encode the nucleocapsid protein (NP), the envelope glycoproteins (Gn and Gc) and an RNA-dependent RNA polymerase (L), respectively. During the viral cycle, NP and L drive the processes of transcription (mRNA synthesis) and replication (synthesis of genomic RNA) that occur in the cytoplasm. Thus, targeting these proteins is likely an accurate strategy to inhibit the viral replication.
Currently, there are neither vaccines nor effective therapies to treat bunyavirus infection in humans. Ribavirin, however, has been shown to inhibit CCHFV replication in Vero cells, reducing the mean time to death in infant mice and partially protecting infected STAT-1 KO mice. Several studies reported the efficiency of oral or intravenous use of ribavirin to treat CCHFV infection cases but to date, no double-blind trial had been carried out. Therefore, it is important to initiate research programs aimed towards the development of new medical countermeasures against CCHFV.
Since its discovery in 1998, RNA interference (RNAi) has been successfully applied as a technology to inhibit gene expression. Small interfering RNAs (siRNAs), the mediators of RNAi, are a class of double-stranded RNA molecules (20-25 nucleotides in length) that interfere with translation by inducing sequence-specific degradation of homologous mRNA. Recently, several RNAi-based applications for gene silencing have been developed to target pathogenic human viruses causing acute or chronic infections including HIV-1, influenza virus, respiratory syncytial virus, hepatitis B and C viruses, as well as Marburg and Ebola filoviruses [38-40].
In this report, we tested various chemically synthesized siRNAs for their ability to inhibit HAZV replication in cell culture. We demonstrated that siRNAs targeting the NP mRNA depicted a stronger antiviral effect than those designed to inhibit the L and M segment encoded mRNAs. siRNAs were efficient when transfected in cells before or after HAZV infection and their use in combination with ribavirin induced a synergistic or an additive antiviral effect, according to the dose of ribavirin used. Thus, our study highlights the potential of RNAi in the antiviral treatment of nairovirus infection.
Cell lines and viruses
A549 cells (human lung carcinoma cell line, ATCC CCL-185) and Vero E6 cells (African green monkey kidney, ATCC CRL-1586) were grown in F12K medium and Dulbecco’s modified eagle medium DMEM (Gibco, Invitrogen Corporation, Paisley, United Kingdom), respectively, supplemented with 10% heat-inactivated fetal calf serum (FCS; Invitrogen, Sao Paulo, Brazil) and maintained at 37°C in a 5% CO2 atmosphere. BHK21 cells (baby hamster kidney, ATCC CCL10) were cultured in Glasgow minimum essential medium GMEM (Gibco, Invitrogen Corporation) with 10% FCS, 10% tryptose phosphate (Sigma-Aldrich, St Quentin-Fallavier, France) and 50 mM HEPES (Gibco, Invitrogen Corporation) at 37°C in a 5% CO2 atmosphere.
The HAZV strain JC280 used in these experiments was produced by infecting 90% confluent BHK21 cells at a multiplicity of infection (MOI) of 0.001. Virus was titrated using a focus-forming assay in Vero E6 cells as described below. Viral stocks usually reached 106-107 foci forming units per ml (ffu/ml).
Inhibition of HAZV replication using segment-specific siRNAs
To evaluate the inhibitory activity of siRNAs on HAZV replication, twelve siRNAs were designed to target the mRNAs produced by the L, M, and S genomic segments. We analyzed the inhibitory effect of the L, M and S-specific siRNAs. As shown in figure 1A, when used at 100 nM, siS1, siS2 and siS3 strongly inhibited virus replication by 87.8%, 91.6% and 86.1%, respectively, compared to siNT control (p < 0.05). In contrast, siS4 did not induce any significant antiviral effect in A549-infected cells. siM1, siM2 and siM3 showed moderate but significant activities on HAZV replication (38.3%, 56.2% and 29.4% of inhibition respectively) whereas siM4 did not have any activity (figure 1B). Lastly, siL2, siL3 and siL4 did not inhibit HAZV replication (p > 0.05) while siL1 slightly did.
Inhibition of HAZV replication by siS1 and siS2 in A549 cells
Since siS1 and siS2 showed the most efficient anti HAZV activity, we decided to further focus on the characterization of these two siRNAs. We first tested their efficacy at 100 nM in cells infected at different multiplicity of infection (i.e. MOI 0.01, 0.1 and 1). Whatever the viral load, a significant inhibition of HAZV replication was observed for both siRNAs when compared to siNT: the inhibition ranged from 83.1% to 97.1% for siS1 and from 94.4% to 98.3% for siS2.
Because siRNAs treatment could be cytotoxic and therefore may affect viral growth, we performed a Trypan blue exclusion assay to evaluate cell growth and viability upon siRNA transfection. The morphology of treated cells was examined daily using phase-contrast light microscopy. Three days post siRNA transfections, the cell number per well was determined and compared to non treated cells by manual counting with a hematocytometer. In these experiments, we did not detect any cytotoxic effect of siS1 and siS2 on A549 cells at any concentrations used (not shown). We also investigated whether the IFN pathway could be stimulated in siRNAs transfected cells as reported in earlier studies. We found that the siRNAs tested did not induce any IFN-β response whereas the poly(I:C) control was a good stimulator (not shown). Thus, HAZV inhibition was siS1 and siS2 specific and was not due to any side effects.
We then examined the antiviral effects of increasing concentrations of siS1 and siS2. A concentration dependent inhibitory activity was observed for both siRNAs when compared to siNT. siS1 induced an average inhibition of 64.2% at 0.1 nM and 86% at 100 nM. The production of infectious HAZV particles was reduced by 70.6% at 0.1 nM and 94% at 100 nM when cells were transfected with siS2. No significant viral inhibition was observed at siRNAs concentration of 0.01 nM. From these experiments the estimated 50% effective concentration (EC50) was 0.09 nM for siS1 and 0.07 nM for siS2. Thus, both siRNAs efficiently inhibited HAZV replication in a concentration dependant manner, in cell culture.