Ribavirin Had Demonstrable Effects on the Crimean-Congo Hemorrhagic Fever Virus (CCHFV) Population and Load in a Patient With CCHF Infection

Ribavirin Had Demonstrable Effects on the Crimean-Congo Hemorrhagic Fever Virus (CCHFV)... Abstract The use of ribavirin to treat Crimean-Congo hemorrhagic fever virus (CCHFV) infection has been controversial, based on uncertainties about its antiviral efficacy in clinical case studies. We studied the effect of ribavirin treatment on viral populations in a recent case by deep-sequencing analysis of plasma samples obtained from a CCHFV-infected patient before, during, and after a 5-day regimen of ribavirin treatment. The CCHFV load dropped during ribavirin treatment, and subclonal diversity (transitions) and indels increased in viral genomes during treatment. Although the results are based on a single case, these data demonstrate the mutagenic effect of ribavirin on CCHFV in vivo. Crimean-Congo hemorrhagic fever virus, ribavirin, viral genomics Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that causes Crimean-Congo hemorrhagic fever. CCHFV is a negative-stranded, segmented RNA virus in the genus Orthonairovirus, family Nairoviridae. CCHFV infections have been observed in Europe, Asia, and Africa, but treatment options for patients are limited. Supportive care is the most common form of treatment. Ribavirin has also been used for treatment and prophylaxis, although its efficacy remains controversial [1, 2]. Recently, 2 autochthonous cases of Crimean-Congo hemorrhagic fever were observed in Spain [3]. The secondary case was treated with ribavirin for 5 days, which allowed us to measure virologic response to treatment by measuring the ribavirin-mediated mutagenic activity on the viral population. METHOD Sample Collection and Processing The epidemiologic characteristics and clinical presentation of the CCHFV-infected secondary case presented here has been described previously [3]. Five days into the course of infection, the patient received 1000 mg of ribavirin orally every 6 hours for 24 hours and intravenously for the next 24 hours, followed by 500 mg of intravenous ribavirin every 8 hours for 4 days, in addition to supportive care (Figure 1A). Plasma samples were collected during the course of treatment and used for deep-sequencing and genomic analyses. Viral loads were measured by quantitative real-time reverse transcription–polymerase chain reaction (PCR) analysis. Figure 1. View largeDownload slide Crimean-Congo hemorrhagic fever virus (CCHFV) genomic characteristics. A, Treatment regimen of the second patient with Crimean-Congo hemorrhagic fever, where days are numbered according to length of time after the initiation of ribavirin treatment on day 1. B, CCHFV load (right y-axis), measured on the basis of CCHFV RNA detection by reverse transcription–polymerase chain reaction, decreased during ribavirin treatment (x-axis). Viral populations were enriched by RNA Access, using CCHFV-specific oligomeric probes; sequenced on an Illumina MiSeq; and aligned to the consensus sequence of the day 1 sample. The percentage coverage of sequenced segments (S, M, and L; left y-axis) varied with the viral load. C, Paired-end FASTQ reads from each sample that aligned to the CCHFV consensus sequence were segregated on the basis of the strandedness of the input sample RNA. Values are percentages of reads that are negative stranded (left y-axis). D, Subclonal diversity (left y-axis), measured on the basis of the number of base changes per site per genome, in CCHFV populations increased during ribavirin treatment. Values are measured with respect to day 1 and were derived from base changes with a frequency ≥2% at each site. Subclonal diversity was significantly different across sample days (P = .0336, by 2-way analysis of variance). Closed circles indicate samples collected during ribavirin treatment. Open circles indicate samples collected after the cessation of ribavirin treatment. Figure 1. View largeDownload slide Crimean-Congo hemorrhagic fever virus (CCHFV) genomic characteristics. A, Treatment regimen of the second patient with Crimean-Congo hemorrhagic fever, where days are numbered according to length of time after the initiation of ribavirin treatment on day 1. B, CCHFV load (right y-axis), measured on the basis of CCHFV RNA detection by reverse transcription–polymerase chain reaction, decreased during ribavirin treatment (x-axis). Viral populations were enriched by RNA Access, using CCHFV-specific oligomeric probes; sequenced on an Illumina MiSeq; and aligned to the consensus sequence of the day 1 sample. The percentage coverage of sequenced segments (S, M, and L; left y-axis) varied with the viral load. C, Paired-end FASTQ reads from each sample that aligned to the CCHFV consensus sequence were segregated on the basis of the strandedness of the input sample RNA. Values are percentages of reads that are negative stranded (left y-axis). D, Subclonal diversity (left y-axis), measured on the basis of the number of base changes per site per genome, in CCHFV populations increased during ribavirin treatment. Values are measured with respect to day 1 and were derived from base changes with a frequency ≥2% at each site. Subclonal diversity was significantly different across sample days (P = .0336, by 2-way analysis of variance). Closed circles indicate samples collected during ribavirin treatment. Open circles indicate samples collected after the cessation of ribavirin treatment. The research protocol was approved by the relevant institutional ethics committees, and all human participants gave written informed consent. CCHFV Sequencing RNA was extracted from the patient’s plasma samples, using the QIAamp Viral RNA Mini Kit (Qiagen), following the manufacturer’s instructions. Ribosomal RNA and carrier RNA were depleted with specific probes and Oligo(dT)20 (Invitrogen), respectively [4]. Briefly, the ribosomal RNA probe mix and the Oligo(dT)20 were hybridized to the total RNA and treated with 10 units of Hybridase Thermostable RNase H (Epicentre). After the RNase reaction, RNA was cleaned with 2.2X RNA AMPure XP magnetic beads (Agencourt), treated with 2 units of Turbo DNase I (ThermoFisher Scientific), and cleaned a second time with 2.2X RNA magnetic beads. Depleted RNA was resuspended in 10 µL of nuclease-free water. CCHFV sequencing from both depleted and nondepleted RNA aliquots was performed using a targeted enrichment approach as previously described [5]. A set of 478 unique 80mer probes tiled along the genome sequence obtained from the patient sample collected on 31 August, immediately before ribavirin treatment was commenced, was synthesized and used as baits for target enrichment [6]. The sample from 31 August is referred to as sample zero. Samples were barcoded with nonoverlapping dual indexes, pooled, and sequenced using the MiSeq Reagent kit v3 (Illumina) on an Illumina MiSeq instrument with a minimum of 2 × 151-bp reads. Alignment files for all the libraries assessed here are available at Bioproject PRJNA417384 and National Center for Biotechnology Information Sequence Read Archive project number SRP124456. CCHFV Genomic Analysis For analysis of the dual-indexed reads, paired-ended FASTQs were analyzed using a validated analysis pipeline (VSALIGN) [7]. VSALIGN is built on Perl and uses the open-source programs Cutadapt and Prinseq-lite for preprocessing of samples, including adapter removal, PCR duplicate removal, and quality filtering of the index (<30 Phred) and reads (<20 Phred). Additional preprocessing steps are also included to remove chimeric sequences, reads with bad or no mate, and reads that do not have significant matches to the reference sequence. For this purpose, we use as a reference the consensus sequence of CCHFV obtained 2 hours before initiation of ribavirin treatment (Day 1). Sequences were aligned to the reference sequence by using default parameters in VSALIGN to determine the frequency of single-nucleotide polymorphisms (SNPs). Subclonal diversity and the frequency of SNPs, insertions and deletions (indels), transitions, and transversions were measured for nucleotide positions meeting the minimum depth of 200 reads, or >2% of the population. Two-way analyses of variance (ANOVAs) between sample collection days and subclonal diversity values were performed using GraphPad Prism, version 7.03 for Windows (La Jolla, CA). RESULTS CCHFV Population Characteristics Coverage from plasma samples ranged from 2%–99%, in direct relation to the previously reported viral load (Figure 1B). Overall coverage decreased dramatically during ribavirin treatment. Sequence coverage was relatively equal in the S and L segments across samples, while the M segment displayed lower coverage. Lower coverage of the M segment could be attributed to lower efficiency of the CCHFV-specific M probes or to a differing proportion of negatively stranded M segments in the sample (Figure 1C), although both explanations are unconvincing. Since sequence coverage is related to total viral RNA (as estimated by read depth), a more likely reason for lower coverage of the M segment at days 2 and 3 is the accumulation of defective interfering particles of CCHFVs that lack the M segment, as has been previously described [8]. The day 4 sample resulted in coverage of only 0%–2.4% of the viral genome and was therefore excluded from further analysis. To quantify the effect of ribavirin on CCHFV intrahost variation, subclonal diversity was measured for each sample collected during and after ribavirin treatment (Figure 1D). Subclonal diversity estimates the rate of SNPs, insertions, and deletions that occurred between the sample sequence and the reference sequence. The value is a function of genome coverage and is normalized by depth at each position. Subclonal diversity increased in the CCHFV M and L segments during ribavirin treatment and plateaued after the cessation of treatment. Few indels appeared to be shared across sample days, and there was a significant statistical difference between indel frequencies between collection days (P = .0336, by 2-way ANOVA). Subclonal diversity in S also increased slightly during treatment days and then varied stochastically in subsequent days. The changes in subclonal diversity were also correlated with the changes in viral load (Figure 1B). Subclonal Analysis of CCHFV Populations The mutational rate of RNA viruses has been determined to be approximately 10–5 bases per site per genome, with random low-frequency base changes occurring throughout the viral genome and transitions more common than transversions [9]. Ribavirin treatment of poliovirus and hepatitis C virus resulted in an increase in G-to-A and C-to-T transitions, as well as an increase, although less so, in A-to-G and T-to-C transitions, compared with the presumed natural mutational rate [10, 11]. CCHFV sequencing results during and after ribavirin treatment showed a similar random distribution of SNPs along the viral genome (Figure 2A). The viral population from day 3 of ribavirin treatment displayed a higher frequency of SNPs as compared to viruses from samples collected the day before or the day after the termination of treatment. Low-frequency SNPs (2%–10%) accumulated in all viral segments, while high-frequency SNPs (>10%) were observed primarily in the L segment. High-frequency SNPs were not shared across samples collected on different days, suggesting that viruses with mutated genomes did not persist in the patient over time. Figure 2. View largeDownload slide Single-nucleotide polymorphisms (SNPs) and indels in the Crimean-Congo hemorrhagic fever virus (CCHFV) population during and after ribavirin treatment. A, SNP frequencies (left y-axis) across each segment (x-axis) display intrahost viral diversity. Each data point is colored according to each sample in which the SNP frequency was ≥2% at each position. The boxes above the graphs represent open-reading frames corresponding to the nucleoprotein (NP), mucin, GP38 (38), Gn, Nsm (N), Gc, and RdRP domains. B, Rates of transitions (trs), transversions (trv), and indels per site per genome (left y-axis) were measured for each sample (x-axis). The indels were significantly accumulated in the L segment (P = .0202, by 2-way analysis of variance). Viral load was mapped on the right y-axis as a reference. Closed circles indicate samples collected during ribavirin treatment. Open circles indicate samples collected after the cessation of ribavirin treatment. C, The frequency of base changes in samples, by day after treatment initiation. The total number of positions analyzed involve 854 nucleotides distributed across the L segment. Values are the sum of base changes per site per genome for positions where the SNP frequency was ≥2%. Figure 2. View largeDownload slide Single-nucleotide polymorphisms (SNPs) and indels in the Crimean-Congo hemorrhagic fever virus (CCHFV) population during and after ribavirin treatment. A, SNP frequencies (left y-axis) across each segment (x-axis) display intrahost viral diversity. Each data point is colored according to each sample in which the SNP frequency was ≥2% at each position. The boxes above the graphs represent open-reading frames corresponding to the nucleoprotein (NP), mucin, GP38 (38), Gn, Nsm (N), Gc, and RdRP domains. B, Rates of transitions (trs), transversions (trv), and indels per site per genome (left y-axis) were measured for each sample (x-axis). The indels were significantly accumulated in the L segment (P = .0202, by 2-way analysis of variance). Viral load was mapped on the right y-axis as a reference. Closed circles indicate samples collected during ribavirin treatment. Open circles indicate samples collected after the cessation of ribavirin treatment. C, The frequency of base changes in samples, by day after treatment initiation. The total number of positions analyzed involve 854 nucleotides distributed across the L segment. Values are the sum of base changes per site per genome for positions where the SNP frequency was ≥2%. To define the type of mutations occurring in each sample during and after ribavirin treatment, the frequency of indels, transitions, and transversions was measured over time (Figure 2B). The frequency of transitions and, to a lesser degree, transversions increased between day 2 and day 3 of ribavirin treatment. The nucleotide diversity further increased by day 6 after the cessation of treatment and plateaued in the subsequent days. Indels displayed a statistically significant rate of change in the CCHFV L segment (P = .0202, by 2-way ANOVA); the indel frequency increased dramatically between days 2 and 3 of ribavirin treatment and remained high until day 8. These indels were observed to primarily be insertions at G and C positions (data not shown). To define whether the subclonal diversity resembled the characteristic base changes observed during ribavirin treatment of other viral infections, the frequency of individual base changes was measured (Figure 2C). We restricted our analysis to nucleotide positions in the CCHFV L segment, which had coverage data across all samples (854 nucleotide positions), to avoid the inclusion of regions with low coverage. The sample collected on day 9 was excluded from this analysis because only 13% of the L segment was recovered, thus narrowing the regions with coverage across all samples. Results of this analysis identified an increased rate of T-to-C transitions, to 10–3 changes per site per genome, between days 2 and 3 of ribavirin treatment. The rate of T-to-C transitions then decreased to 0 in samples collected after the cessation of treatment. Further, the rate of G-to-A and C-to-T changes increased to approximately 10–4 changes per site per genome between days 2 and 3 of ribavirin treatment and changed little from this value in viral populations sequenced after ribavirin treatment. Other individual base changes displayed no demonstrable longitudinal pattern. Discussion Nucleoside analogues are a class of drugs that have been used historically to treat hemorrhagic infections, and some of them are currently being developed as candidate drugs for therapeutic use in emerging infections [12]. Ribavirin is a purine nucleoside analogue compound with known antiviral activity against many positive- and negative-stranded RNA viruses in vitro and in vivo. Of the number of proposed mechanisms of action, ribavirin and other nucleoside analogues have a direct antiviral activity that disrupts RNA synthesis by competing with cellular nucleotides, resulting in termination of the polymerizing RNA chains or mismatching of bases that lead to the increased accumulation of mutations throughout the viral genome [10]. Each nucleoside analogue that induces mutagenesis therefore leaves a “footprint” in the viral genome. Studies measuring the mutational effect of ribavirin on viral populations have identified an increase in G-to-A and C-to-T transitions and, in some cases, indels, directly linking this to antiviral activity [11, 13]. Ribavirin also demonstrates antiviral activity against CCHFV in in vivo assays in rodent models [14]. Despite these findings, systematic reviews of studies measuring the effect of ribavirin on CCHFV-infected patient outcomes have been incomplete or inconclusive, leading the field to doubt ribavirin’s efficacy for human treatment [1, 2, 15]. The data obtained from the ribavirin-treated secondary Crimean-Congo hemorrhagic fever case in Spain represent a unique opportunity to characterize the mutagenic “footprint” induced by ribavirin activity against CCHFV in vivo. Our data are compatible with ribavirin producing a measurable effect in viral populations during treatment, inducing increased mutagenesis that correlated with a decrease in the viral titer. We also observed (1) an increase in the rate of subclonal diversity in the viral populations, primarily sustained by T-to-C, G-to-A, and C-to-T transitions and indels in the L segment after 3 days of treatment; (2) that the rates of these mutations remained the same or declined after the cessation of ribavirin treatment and that high-frequency mutations did not persist in the viral population in subsequent days of infection, suggesting that these mutations were unstable or deleterious; and (3) that the rate of mutations in the ribavirin-treated CCHFVs was substantially higher than the fidelity of an RNA polymerase (approximately 10–5 mutations per site per genome). This study analyzed samples collected longitudinally from a single Crimean-Congo hemorrhagic fever case in Spain. Obviously, a single-case study limits significantly our ability to generalize these results. However, CCHFV is an emerging pathogen of global health importance, and the paucity of clinical data on the efficacy of ribavirin as a viable treatment for Crimean-Congo hemorrhagic fever makes these observations valuable. The results support the continued evaluation of ribavirin as an antiviral for use in CCHFV-infected patients and emphasize the need for a randomized control trial that includes the study of viral populations before, during, and after treatment to conclusively determine whether ribavirin’s antiviral effect influences treatment outcomes and the appropriate dosing regimen required for therapeutic use. Notes Acknowledgments. N. E., U. P.-S., E. R. de. A., A. N., M. R. W., M. P. S.-S., and G. P. designed the study. E. R. de. A., A. N., and M. P. S.-S. made the Crimean-Congo hemorrhagic fever diagnosis and determined the viral load and the complete genome of CCHFV from sample zero (the reference sequence of the study). M. D. M. participated on clinical management of the patient with Crimean-Congo hemorrhagic fever and in use of ribavirin to treat Crimean-Congo hemorrhagic fever. U. P.-S., E. R. de. A., A. N., and M. P. S.-S. performed CCHFV sequencing. N. E. performed data analysis. N. E., U. P.-S., and G. P. wrote the manuscript. G. P. and M. R. W. provided editorial oversight. Funding for the study was acquired by M. P. S.-S. and G. P. Oversight of the study was performed by M. P. S.-S., S. B., and G. P.. Disclaimer. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army or the Centers for Disease Control and Prevention, Department of Health and Human Services. Financial support. This work was supported by Red de Investigación Cooperativa en Enfermedades Tropicales (grant RD12/0018/0023); the Efficient Response to Highly Dangerous and Emerging Pathogens at the European Union (EU) Level, a joint action funded under the third EU Health Program (677066); and the Joint Science and Technology Office for Chemical and Biological Defense, Defense Threat Reduction Agency (plan CB10246). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Soares-Weiser K , Thomas S , Thomson G , Garner P . Ribavirin for Crimean-Congo hemorrhagic fever: systematic review and meta-analysis . BMC Infect Dis 2010 ; 10 : 207 . Google Scholar CrossRef Search ADS PubMed 2. Keshtkar-Jahromi M , Kuhn JH , Christova I , Bradfute SB , Jahrling PB , Bavari S . Crimean-Congo hemorrhagic fever: current and future prospects of vaccines and therapies . Antiviral Res 2011 ; 90 : 85 – 92 . Google Scholar CrossRef Search ADS PubMed 3. Negredo A , de la Calle-Prieto F , Palencia-Herrejon E , et al. Autochthonous Crimean-Congo Hemorrhagic Fever in Spain . N Engl J Med 2017 ; 377 : 154 – 61 . Google Scholar CrossRef Search ADS PubMed 4. Morlan JD , Qu K , Sinicropi DV . Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue . PLoS One 2012 ; 7 : e42882 . Google Scholar CrossRef Search ADS PubMed 5. Mate SE , Kugelman JR , Nyenswah TG , et al. Molecular evidence of sexual transmission of ebola virus . N Engl J Med 2015 ; 373 : 2448 – 54 . Google Scholar CrossRef Search ADS PubMed 6. Ramírez de Arellano E , Goyanes MJ , Arsuaga M , et al. Phylogenetic characterization of Crimean-Congo hemorrhagic fever virus, Spain . Emerg Infect Dis 2017 ; 23 : 2078 – 80 . Google Scholar CrossRef Search ADS PubMed 7. Kugelman JR , Wiley MR , Nagle ER , et al. Error baseline rates of five sample preparation methods used to characterize RNA virus populations . PLoS One 2017 ; 12 : e0171333 . Google Scholar CrossRef Search ADS PubMed 8. Weidmann M , Sall AA , Manuguerra JC , et al. Quantitative analysis of particles, genomes and infectious particles in supernatants of haemorrhagic fever virus cell cultures . Virol J 2011 ; 8 : 81 . Google Scholar CrossRef Search ADS PubMed 9. Acevedo A , Brodsky L , Andino R . Mutational and fitness landscapes of an RNA virus revealed through population sequencing . Nature 2014 ; 505 : 686 – 90 . Google Scholar CrossRef Search ADS PubMed 10. Crotty S , Cameron CE , Andino R . RNA virus error catastrophe: direct molecular test by using ribavirin . Proc Natl Acad Sci U S A 2001 ; 98 : 6895 – 900 . Google Scholar CrossRef Search ADS PubMed 11. Dietz J , Schelhorn SE , Fitting D , et al. Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1-infected patients . J Virol 2013 ; 87 : 6172 – 81 . Google Scholar CrossRef Search ADS PubMed 12. Warren TK , Jordan R , Lo MK , et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys . Nature 2016 ; 531 : 381 – 5 . Google Scholar CrossRef Search ADS PubMed 13. Pauly MD , Lauring AS . Effective lethal mutagenesis of influenza virus by three nucleoside analogs . J Virol 2015 ; 89 : 3584 – 97 . Google Scholar CrossRef Search ADS PubMed 14. Oestereich L , Rieger T , Neumann M , et al. Evaluation of antiviral efficacy of ribavirin, arbidol, and T-705 (favipiravir) in a mouse model for Crimean-Congo hemorrhagic fever . PLoS Negl Trop Dis 2014 ; 8 : e2804 . Google Scholar CrossRef Search ADS PubMed 15. Ascioglu S , Leblebicioglu H , Vahaboglu H , Chan KA . Ribavirin for patients with Crimean-Congo haemorrhagic fever: a systematic review and meta-analysis . J Antimicrob Chemother 2011 ; 66 : 1215 – 22 . Google Scholar CrossRef Search ADS PubMed Published by Oxford University Press for the Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Ribavirin Had Demonstrable Effects on the Crimean-Congo Hemorrhagic Fever Virus (CCHFV) Population and Load in a Patient With CCHF Infection

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Published by Oxford University Press for the Infectious Diseases Society of America 2018.
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0022-1899
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Abstract

Abstract The use of ribavirin to treat Crimean-Congo hemorrhagic fever virus (CCHFV) infection has been controversial, based on uncertainties about its antiviral efficacy in clinical case studies. We studied the effect of ribavirin treatment on viral populations in a recent case by deep-sequencing analysis of plasma samples obtained from a CCHFV-infected patient before, during, and after a 5-day regimen of ribavirin treatment. The CCHFV load dropped during ribavirin treatment, and subclonal diversity (transitions) and indels increased in viral genomes during treatment. Although the results are based on a single case, these data demonstrate the mutagenic effect of ribavirin on CCHFV in vivo. Crimean-Congo hemorrhagic fever virus, ribavirin, viral genomics Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that causes Crimean-Congo hemorrhagic fever. CCHFV is a negative-stranded, segmented RNA virus in the genus Orthonairovirus, family Nairoviridae. CCHFV infections have been observed in Europe, Asia, and Africa, but treatment options for patients are limited. Supportive care is the most common form of treatment. Ribavirin has also been used for treatment and prophylaxis, although its efficacy remains controversial [1, 2]. Recently, 2 autochthonous cases of Crimean-Congo hemorrhagic fever were observed in Spain [3]. The secondary case was treated with ribavirin for 5 days, which allowed us to measure virologic response to treatment by measuring the ribavirin-mediated mutagenic activity on the viral population. METHOD Sample Collection and Processing The epidemiologic characteristics and clinical presentation of the CCHFV-infected secondary case presented here has been described previously [3]. Five days into the course of infection, the patient received 1000 mg of ribavirin orally every 6 hours for 24 hours and intravenously for the next 24 hours, followed by 500 mg of intravenous ribavirin every 8 hours for 4 days, in addition to supportive care (Figure 1A). Plasma samples were collected during the course of treatment and used for deep-sequencing and genomic analyses. Viral loads were measured by quantitative real-time reverse transcription–polymerase chain reaction (PCR) analysis. Figure 1. View largeDownload slide Crimean-Congo hemorrhagic fever virus (CCHFV) genomic characteristics. A, Treatment regimen of the second patient with Crimean-Congo hemorrhagic fever, where days are numbered according to length of time after the initiation of ribavirin treatment on day 1. B, CCHFV load (right y-axis), measured on the basis of CCHFV RNA detection by reverse transcription–polymerase chain reaction, decreased during ribavirin treatment (x-axis). Viral populations were enriched by RNA Access, using CCHFV-specific oligomeric probes; sequenced on an Illumina MiSeq; and aligned to the consensus sequence of the day 1 sample. The percentage coverage of sequenced segments (S, M, and L; left y-axis) varied with the viral load. C, Paired-end FASTQ reads from each sample that aligned to the CCHFV consensus sequence were segregated on the basis of the strandedness of the input sample RNA. Values are percentages of reads that are negative stranded (left y-axis). D, Subclonal diversity (left y-axis), measured on the basis of the number of base changes per site per genome, in CCHFV populations increased during ribavirin treatment. Values are measured with respect to day 1 and were derived from base changes with a frequency ≥2% at each site. Subclonal diversity was significantly different across sample days (P = .0336, by 2-way analysis of variance). Closed circles indicate samples collected during ribavirin treatment. Open circles indicate samples collected after the cessation of ribavirin treatment. Figure 1. View largeDownload slide Crimean-Congo hemorrhagic fever virus (CCHFV) genomic characteristics. A, Treatment regimen of the second patient with Crimean-Congo hemorrhagic fever, where days are numbered according to length of time after the initiation of ribavirin treatment on day 1. B, CCHFV load (right y-axis), measured on the basis of CCHFV RNA detection by reverse transcription–polymerase chain reaction, decreased during ribavirin treatment (x-axis). Viral populations were enriched by RNA Access, using CCHFV-specific oligomeric probes; sequenced on an Illumina MiSeq; and aligned to the consensus sequence of the day 1 sample. The percentage coverage of sequenced segments (S, M, and L; left y-axis) varied with the viral load. C, Paired-end FASTQ reads from each sample that aligned to the CCHFV consensus sequence were segregated on the basis of the strandedness of the input sample RNA. Values are percentages of reads that are negative stranded (left y-axis). D, Subclonal diversity (left y-axis), measured on the basis of the number of base changes per site per genome, in CCHFV populations increased during ribavirin treatment. Values are measured with respect to day 1 and were derived from base changes with a frequency ≥2% at each site. Subclonal diversity was significantly different across sample days (P = .0336, by 2-way analysis of variance). Closed circles indicate samples collected during ribavirin treatment. Open circles indicate samples collected after the cessation of ribavirin treatment. The research protocol was approved by the relevant institutional ethics committees, and all human participants gave written informed consent. CCHFV Sequencing RNA was extracted from the patient’s plasma samples, using the QIAamp Viral RNA Mini Kit (Qiagen), following the manufacturer’s instructions. Ribosomal RNA and carrier RNA were depleted with specific probes and Oligo(dT)20 (Invitrogen), respectively [4]. Briefly, the ribosomal RNA probe mix and the Oligo(dT)20 were hybridized to the total RNA and treated with 10 units of Hybridase Thermostable RNase H (Epicentre). After the RNase reaction, RNA was cleaned with 2.2X RNA AMPure XP magnetic beads (Agencourt), treated with 2 units of Turbo DNase I (ThermoFisher Scientific), and cleaned a second time with 2.2X RNA magnetic beads. Depleted RNA was resuspended in 10 µL of nuclease-free water. CCHFV sequencing from both depleted and nondepleted RNA aliquots was performed using a targeted enrichment approach as previously described [5]. A set of 478 unique 80mer probes tiled along the genome sequence obtained from the patient sample collected on 31 August, immediately before ribavirin treatment was commenced, was synthesized and used as baits for target enrichment [6]. The sample from 31 August is referred to as sample zero. Samples were barcoded with nonoverlapping dual indexes, pooled, and sequenced using the MiSeq Reagent kit v3 (Illumina) on an Illumina MiSeq instrument with a minimum of 2 × 151-bp reads. Alignment files for all the libraries assessed here are available at Bioproject PRJNA417384 and National Center for Biotechnology Information Sequence Read Archive project number SRP124456. CCHFV Genomic Analysis For analysis of the dual-indexed reads, paired-ended FASTQs were analyzed using a validated analysis pipeline (VSALIGN) [7]. VSALIGN is built on Perl and uses the open-source programs Cutadapt and Prinseq-lite for preprocessing of samples, including adapter removal, PCR duplicate removal, and quality filtering of the index (<30 Phred) and reads (<20 Phred). Additional preprocessing steps are also included to remove chimeric sequences, reads with bad or no mate, and reads that do not have significant matches to the reference sequence. For this purpose, we use as a reference the consensus sequence of CCHFV obtained 2 hours before initiation of ribavirin treatment (Day 1). Sequences were aligned to the reference sequence by using default parameters in VSALIGN to determine the frequency of single-nucleotide polymorphisms (SNPs). Subclonal diversity and the frequency of SNPs, insertions and deletions (indels), transitions, and transversions were measured for nucleotide positions meeting the minimum depth of 200 reads, or >2% of the population. Two-way analyses of variance (ANOVAs) between sample collection days and subclonal diversity values were performed using GraphPad Prism, version 7.03 for Windows (La Jolla, CA). RESULTS CCHFV Population Characteristics Coverage from plasma samples ranged from 2%–99%, in direct relation to the previously reported viral load (Figure 1B). Overall coverage decreased dramatically during ribavirin treatment. Sequence coverage was relatively equal in the S and L segments across samples, while the M segment displayed lower coverage. Lower coverage of the M segment could be attributed to lower efficiency of the CCHFV-specific M probes or to a differing proportion of negatively stranded M segments in the sample (Figure 1C), although both explanations are unconvincing. Since sequence coverage is related to total viral RNA (as estimated by read depth), a more likely reason for lower coverage of the M segment at days 2 and 3 is the accumulation of defective interfering particles of CCHFVs that lack the M segment, as has been previously described [8]. The day 4 sample resulted in coverage of only 0%–2.4% of the viral genome and was therefore excluded from further analysis. To quantify the effect of ribavirin on CCHFV intrahost variation, subclonal diversity was measured for each sample collected during and after ribavirin treatment (Figure 1D). Subclonal diversity estimates the rate of SNPs, insertions, and deletions that occurred between the sample sequence and the reference sequence. The value is a function of genome coverage and is normalized by depth at each position. Subclonal diversity increased in the CCHFV M and L segments during ribavirin treatment and plateaued after the cessation of treatment. Few indels appeared to be shared across sample days, and there was a significant statistical difference between indel frequencies between collection days (P = .0336, by 2-way ANOVA). Subclonal diversity in S also increased slightly during treatment days and then varied stochastically in subsequent days. The changes in subclonal diversity were also correlated with the changes in viral load (Figure 1B). Subclonal Analysis of CCHFV Populations The mutational rate of RNA viruses has been determined to be approximately 10–5 bases per site per genome, with random low-frequency base changes occurring throughout the viral genome and transitions more common than transversions [9]. Ribavirin treatment of poliovirus and hepatitis C virus resulted in an increase in G-to-A and C-to-T transitions, as well as an increase, although less so, in A-to-G and T-to-C transitions, compared with the presumed natural mutational rate [10, 11]. CCHFV sequencing results during and after ribavirin treatment showed a similar random distribution of SNPs along the viral genome (Figure 2A). The viral population from day 3 of ribavirin treatment displayed a higher frequency of SNPs as compared to viruses from samples collected the day before or the day after the termination of treatment. Low-frequency SNPs (2%–10%) accumulated in all viral segments, while high-frequency SNPs (>10%) were observed primarily in the L segment. High-frequency SNPs were not shared across samples collected on different days, suggesting that viruses with mutated genomes did not persist in the patient over time. Figure 2. View largeDownload slide Single-nucleotide polymorphisms (SNPs) and indels in the Crimean-Congo hemorrhagic fever virus (CCHFV) population during and after ribavirin treatment. A, SNP frequencies (left y-axis) across each segment (x-axis) display intrahost viral diversity. Each data point is colored according to each sample in which the SNP frequency was ≥2% at each position. The boxes above the graphs represent open-reading frames corresponding to the nucleoprotein (NP), mucin, GP38 (38), Gn, Nsm (N), Gc, and RdRP domains. B, Rates of transitions (trs), transversions (trv), and indels per site per genome (left y-axis) were measured for each sample (x-axis). The indels were significantly accumulated in the L segment (P = .0202, by 2-way analysis of variance). Viral load was mapped on the right y-axis as a reference. Closed circles indicate samples collected during ribavirin treatment. Open circles indicate samples collected after the cessation of ribavirin treatment. C, The frequency of base changes in samples, by day after treatment initiation. The total number of positions analyzed involve 854 nucleotides distributed across the L segment. Values are the sum of base changes per site per genome for positions where the SNP frequency was ≥2%. Figure 2. View largeDownload slide Single-nucleotide polymorphisms (SNPs) and indels in the Crimean-Congo hemorrhagic fever virus (CCHFV) population during and after ribavirin treatment. A, SNP frequencies (left y-axis) across each segment (x-axis) display intrahost viral diversity. Each data point is colored according to each sample in which the SNP frequency was ≥2% at each position. The boxes above the graphs represent open-reading frames corresponding to the nucleoprotein (NP), mucin, GP38 (38), Gn, Nsm (N), Gc, and RdRP domains. B, Rates of transitions (trs), transversions (trv), and indels per site per genome (left y-axis) were measured for each sample (x-axis). The indels were significantly accumulated in the L segment (P = .0202, by 2-way analysis of variance). Viral load was mapped on the right y-axis as a reference. Closed circles indicate samples collected during ribavirin treatment. Open circles indicate samples collected after the cessation of ribavirin treatment. C, The frequency of base changes in samples, by day after treatment initiation. The total number of positions analyzed involve 854 nucleotides distributed across the L segment. Values are the sum of base changes per site per genome for positions where the SNP frequency was ≥2%. To define the type of mutations occurring in each sample during and after ribavirin treatment, the frequency of indels, transitions, and transversions was measured over time (Figure 2B). The frequency of transitions and, to a lesser degree, transversions increased between day 2 and day 3 of ribavirin treatment. The nucleotide diversity further increased by day 6 after the cessation of treatment and plateaued in the subsequent days. Indels displayed a statistically significant rate of change in the CCHFV L segment (P = .0202, by 2-way ANOVA); the indel frequency increased dramatically between days 2 and 3 of ribavirin treatment and remained high until day 8. These indels were observed to primarily be insertions at G and C positions (data not shown). To define whether the subclonal diversity resembled the characteristic base changes observed during ribavirin treatment of other viral infections, the frequency of individual base changes was measured (Figure 2C). We restricted our analysis to nucleotide positions in the CCHFV L segment, which had coverage data across all samples (854 nucleotide positions), to avoid the inclusion of regions with low coverage. The sample collected on day 9 was excluded from this analysis because only 13% of the L segment was recovered, thus narrowing the regions with coverage across all samples. Results of this analysis identified an increased rate of T-to-C transitions, to 10–3 changes per site per genome, between days 2 and 3 of ribavirin treatment. The rate of T-to-C transitions then decreased to 0 in samples collected after the cessation of treatment. Further, the rate of G-to-A and C-to-T changes increased to approximately 10–4 changes per site per genome between days 2 and 3 of ribavirin treatment and changed little from this value in viral populations sequenced after ribavirin treatment. Other individual base changes displayed no demonstrable longitudinal pattern. Discussion Nucleoside analogues are a class of drugs that have been used historically to treat hemorrhagic infections, and some of them are currently being developed as candidate drugs for therapeutic use in emerging infections [12]. Ribavirin is a purine nucleoside analogue compound with known antiviral activity against many positive- and negative-stranded RNA viruses in vitro and in vivo. Of the number of proposed mechanisms of action, ribavirin and other nucleoside analogues have a direct antiviral activity that disrupts RNA synthesis by competing with cellular nucleotides, resulting in termination of the polymerizing RNA chains or mismatching of bases that lead to the increased accumulation of mutations throughout the viral genome [10]. Each nucleoside analogue that induces mutagenesis therefore leaves a “footprint” in the viral genome. Studies measuring the mutational effect of ribavirin on viral populations have identified an increase in G-to-A and C-to-T transitions and, in some cases, indels, directly linking this to antiviral activity [11, 13]. Ribavirin also demonstrates antiviral activity against CCHFV in in vivo assays in rodent models [14]. Despite these findings, systematic reviews of studies measuring the effect of ribavirin on CCHFV-infected patient outcomes have been incomplete or inconclusive, leading the field to doubt ribavirin’s efficacy for human treatment [1, 2, 15]. The data obtained from the ribavirin-treated secondary Crimean-Congo hemorrhagic fever case in Spain represent a unique opportunity to characterize the mutagenic “footprint” induced by ribavirin activity against CCHFV in vivo. Our data are compatible with ribavirin producing a measurable effect in viral populations during treatment, inducing increased mutagenesis that correlated with a decrease in the viral titer. We also observed (1) an increase in the rate of subclonal diversity in the viral populations, primarily sustained by T-to-C, G-to-A, and C-to-T transitions and indels in the L segment after 3 days of treatment; (2) that the rates of these mutations remained the same or declined after the cessation of ribavirin treatment and that high-frequency mutations did not persist in the viral population in subsequent days of infection, suggesting that these mutations were unstable or deleterious; and (3) that the rate of mutations in the ribavirin-treated CCHFVs was substantially higher than the fidelity of an RNA polymerase (approximately 10–5 mutations per site per genome). This study analyzed samples collected longitudinally from a single Crimean-Congo hemorrhagic fever case in Spain. Obviously, a single-case study limits significantly our ability to generalize these results. However, CCHFV is an emerging pathogen of global health importance, and the paucity of clinical data on the efficacy of ribavirin as a viable treatment for Crimean-Congo hemorrhagic fever makes these observations valuable. The results support the continued evaluation of ribavirin as an antiviral for use in CCHFV-infected patients and emphasize the need for a randomized control trial that includes the study of viral populations before, during, and after treatment to conclusively determine whether ribavirin’s antiviral effect influences treatment outcomes and the appropriate dosing regimen required for therapeutic use. Notes Acknowledgments. N. E., U. P.-S., E. R. de. A., A. N., M. R. W., M. P. S.-S., and G. P. designed the study. E. R. de. A., A. N., and M. P. S.-S. made the Crimean-Congo hemorrhagic fever diagnosis and determined the viral load and the complete genome of CCHFV from sample zero (the reference sequence of the study). M. D. M. participated on clinical management of the patient with Crimean-Congo hemorrhagic fever and in use of ribavirin to treat Crimean-Congo hemorrhagic fever. U. P.-S., E. R. de. A., A. N., and M. P. S.-S. performed CCHFV sequencing. N. E. performed data analysis. N. E., U. P.-S., and G. P. wrote the manuscript. G. P. and M. R. W. provided editorial oversight. Funding for the study was acquired by M. P. S.-S. and G. P. Oversight of the study was performed by M. P. S.-S., S. B., and G. P.. Disclaimer. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army or the Centers for Disease Control and Prevention, Department of Health and Human Services. Financial support. This work was supported by Red de Investigación Cooperativa en Enfermedades Tropicales (grant RD12/0018/0023); the Efficient Response to Highly Dangerous and Emerging Pathogens at the European Union (EU) Level, a joint action funded under the third EU Health Program (677066); and the Joint Science and Technology Office for Chemical and Biological Defense, Defense Threat Reduction Agency (plan CB10246). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Soares-Weiser K , Thomas S , Thomson G , Garner P . Ribavirin for Crimean-Congo hemorrhagic fever: systematic review and meta-analysis . BMC Infect Dis 2010 ; 10 : 207 . Google Scholar CrossRef Search ADS PubMed 2. Keshtkar-Jahromi M , Kuhn JH , Christova I , Bradfute SB , Jahrling PB , Bavari S . Crimean-Congo hemorrhagic fever: current and future prospects of vaccines and therapies . Antiviral Res 2011 ; 90 : 85 – 92 . Google Scholar CrossRef Search ADS PubMed 3. Negredo A , de la Calle-Prieto F , Palencia-Herrejon E , et al. Autochthonous Crimean-Congo Hemorrhagic Fever in Spain . N Engl J Med 2017 ; 377 : 154 – 61 . Google Scholar CrossRef Search ADS PubMed 4. Morlan JD , Qu K , Sinicropi DV . Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue . PLoS One 2012 ; 7 : e42882 . Google Scholar CrossRef Search ADS PubMed 5. Mate SE , Kugelman JR , Nyenswah TG , et al. Molecular evidence of sexual transmission of ebola virus . N Engl J Med 2015 ; 373 : 2448 – 54 . Google Scholar CrossRef Search ADS PubMed 6. Ramírez de Arellano E , Goyanes MJ , Arsuaga M , et al. Phylogenetic characterization of Crimean-Congo hemorrhagic fever virus, Spain . Emerg Infect Dis 2017 ; 23 : 2078 – 80 . Google Scholar CrossRef Search ADS PubMed 7. Kugelman JR , Wiley MR , Nagle ER , et al. Error baseline rates of five sample preparation methods used to characterize RNA virus populations . PLoS One 2017 ; 12 : e0171333 . Google Scholar CrossRef Search ADS PubMed 8. Weidmann M , Sall AA , Manuguerra JC , et al. Quantitative analysis of particles, genomes and infectious particles in supernatants of haemorrhagic fever virus cell cultures . Virol J 2011 ; 8 : 81 . Google Scholar CrossRef Search ADS PubMed 9. Acevedo A , Brodsky L , Andino R . Mutational and fitness landscapes of an RNA virus revealed through population sequencing . Nature 2014 ; 505 : 686 – 90 . Google Scholar CrossRef Search ADS PubMed 10. Crotty S , Cameron CE , Andino R . RNA virus error catastrophe: direct molecular test by using ribavirin . Proc Natl Acad Sci U S A 2001 ; 98 : 6895 – 900 . Google Scholar CrossRef Search ADS PubMed 11. Dietz J , Schelhorn SE , Fitting D , et al. Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1-infected patients . J Virol 2013 ; 87 : 6172 – 81 . Google Scholar CrossRef Search ADS PubMed 12. Warren TK , Jordan R , Lo MK , et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys . Nature 2016 ; 531 : 381 – 5 . Google Scholar CrossRef Search ADS PubMed 13. Pauly MD , Lauring AS . Effective lethal mutagenesis of influenza virus by three nucleoside analogs . J Virol 2015 ; 89 : 3584 – 97 . Google Scholar CrossRef Search ADS PubMed 14. Oestereich L , Rieger T , Neumann M , et al. Evaluation of antiviral efficacy of ribavirin, arbidol, and T-705 (favipiravir) in a mouse model for Crimean-Congo hemorrhagic fever . PLoS Negl Trop Dis 2014 ; 8 : e2804 . Google Scholar CrossRef Search ADS PubMed 15. Ascioglu S , Leblebicioglu H , Vahaboglu H , Chan KA . Ribavirin for patients with Crimean-Congo haemorrhagic fever: a systematic review and meta-analysis . J Antimicrob Chemother 2011 ; 66 : 1215 – 22 . Google Scholar CrossRef Search ADS PubMed Published by Oxford University Press for the Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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The Journal of Infectious DiseasesOxford University Press

Published: Mar 23, 2018

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