Engineered Orange Ectopically Expressing the Arabidopsis ß-Caryophyllene Synthase Is Not Attractive to Diaphorina citri, the Vector of the Bacterial Pathogen Associated to Huanglongbing.

Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species (Candidatus Liberibacter spp.) or their insect vectors (Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit (E)-ß-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field.

Soil Salinity Limits Plant Shade Avoidance.

Global food production is set to keep increasing despite a predicted decrease in total arable land [1]. To achieve higher production, denser planting will be required on increasingly degraded soils. When grown in dense stands, crops elongate and raise their leaves in an effort to reach sunlight, a process termed shade avoidance [2]. Shade is perceived by a reduction in the ratio of red (R) to far-red (FR) light and results in the stabilization of a class of transcription factors known as PHYTOCHROME INTERACTING FACTORS (PIFs) [3, 4]. PIFs activate the expression of auxin biosynthesis genes [4, 5] and enhance auxin sensitivity [6], which promotes cell-wall loosening and drives elongation growth. Despite our molecular understanding of shade-induced growth, little is known about how this developmental program is integrated with other environmental factors. Here, we demonstrate that low levels of NaCl in soil strongly impair the ability of plants to respond to shade. This block is dependent upon abscisic acid (ABA) signaling and the canonical ABA signaling pathway. Low R:FR light enhances brassinosteroid (BR) signaling through BRASSINOSTEROID SIGNALING KINASE 5 (BSK5) and leads to the activation of BRI1 EMS SUPPRESSOR 1 (BES1). ABA inhibits BSK5 upregulation and interferes with GSK3-like kinase inactivation by the BR pathway, thus leading to a suppression of BES1:PIF function. By demonstrating a link between light, ABA-, and BR-signaling pathways, this study provides an important step forward in our understanding of how multiple environmental cues are integrated into plant development.

Diversity of yeast species from Dutch garden soil and the description of six novel Ascomycetes.

A Citizen Science initiative by the Westerdijk Fungal Biodiversity Institute and the Utrecht University Museum gave rise to a project where fungal and yeast isolates were obtained and identified from Dutch soil samples. During the current study, 386 yeast strains were isolated from 157 different locations in the Netherlands. These strains were identified using sequence data of the large-subunit rRNA gene (D1/D2 region) and the internal transcribed spacer 1 and 2 regions. A total of 53 different yeast species were found as well as 15 potentially novel species. Six novel ascomycetous species are described during this study that include Hanseniaspora mollemarum sp. nov., Ogataea degrootiae sp. nov., Pichia gijzeniarum sp. nov., Saccharomycopsis oosterbeekiorum sp. nov., Trichomonascus vanleenenius sp. nov. and Zygoascus flipseniorum sp. nov. This study made it possible to incorporate numerous yeast isolates into the CBS collection without any restrictions, which make these isolates readily available for use by others. Many of the isolates represented species of which only a few isolates or even only a single ex-type strain were available. Therefore, it is a clear indication that such biodiversity-orientated Citizen Science projects can enrich the pool of available yeasts for future research projects.

A stress recovery signaling network for enhanced flooding tolerance in Arabidopsis thaliana.

Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D, SENESCENCE-ASSOCIATED GENE113, and ORESARA1, which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis.

Forward genetic screens identify a role for the mitochondrial HER2 in E-2-hexenal responsiveness.

KEY MESSAGE: This work adds a new player, HER2, downstream of the perception of E-2-hexenal, a green leaf volatile, and shows that E-2-hexenal specifically changes the redox status of the mitochondria. It is widely accepted that plants produce and respond to green leaf volatiles (GLVs), but the molecular components involved in transducing their perception are largely unknown. The GLV E-2-hexenal inhibits root elongation in seedlings and, using this phenotype, we isolated E-2-hexenal response (her) Arabidopsis thaliana mutants. Using map-based cloning we positioned the her2 mutation to the At5g63620 locus, resulting in a phenylalanine instead of serine on position 223. Knockdown and overexpression lines of HER2 confirmed the role of HER2, which encodes an oxidoreductase, in the responsiveness to E-2-hexenal. Since E-2-hexenal is a reactive electrophile species, which are known to influence the redox status of cells, we utilized redox sensitive GFP2 (roGFP2) to determine the redox status of E-2-hexenal-treated root cells. Since the signal peptide of HER2 directed mCherry to the mitochondria, we targeted the expression of roGFP2 to this organelle besides the cytosol. E-2-hexenal specifically induced a change in the redox status in the mitochondria. We did not see a difference in the redox status in her2 compared to wild-type Arabidopsis. Still, the mitochondrial redox status did not change with Z-3-hexenol, another abundant GLV. These results indicate that HER2 is involved in transducing the perception of E-2-hexenal, which changes the redox status of the mitochondria.

Architecture and Dynamics of the Jasmonic Acid Gene Regulatory Network.

Jasmonic acid (JA) is a critical hormonal regulator of plant growth and defense. To advance our understanding of the architecture and dynamic regulation of the JA gene regulatory network, we performed a high-resolution RNA-seq time series of methyl JA-treated Arabidopsis thaliana at 15 time points over a 16-h period. Computational analysis showed that methyl JA (MeJA) induces a burst of transcriptional activity, generating diverse expression patterns over time that partition into distinct sectors of the JA response targeting specific biological processes. The presence of transcription factor (TF) DNA binding motifs correlated with specific TF activity during temporal MeJA-induced transcriptional reprogramming. Insight into the underlying dynamic transcriptional regulation mechanisms was captured in a chronological model of the JA gene regulatory network. Several TFs, including MYB59 and bHLH27, were uncovered as early network components with a role in pathogen and insect resistance. Analysis of subnetworks surrounding the TFs ORA47, RAP2.6L, MYB59, and ANAC055, using transcriptome profiling of overexpressors and mutants, provided insights into their regulatory role in defined modules of the JA network. Collectively, our work illuminates the complexity of the JA gene regulatory network, pinpoints and validates previously unknown regulators, and provides a valuable resource for functional studies on JA signaling components in plant defense and development.

Arabidopsis JASMONATE-INDUCED OXYGENASES down-regulate plant immunity by hydroxylation and inactivation of the hormone jasmonic acid.

The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JA-responsive gene expression by the bioactive form JA-isoleucine have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. Because they are induced by JA we named them JASMONATE-INDUCED OXYGENASES (JOXs). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in overaccumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses.

Gain and Loss of Floral Scent Production through Changes in Structural Genes during Pollinator-Mediated Speciation.

The interactions of plants with their pollinators are thought to be a driving force in the evolution of angiosperms. Adaptation to a new pollinator involves coordinated changes in multiple floral traits controlled by multiple genes. Surprisingly, such complex genetic shifts have happened numerous times during evolution. Here we report on the genetic basis of the changes in one such trait, floral scent emission, in the genus Petunia (Solanaceae). The increase in the quantity and complexity of the volatiles during the shift from bee to hawkmoth pollination was due to de novo expression of the genes encoding benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) and benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase (BPBT) together with moderately increased transcript levels for most enzymes of the phenylpropanoid/benzenoid pathway. Loss of cinnamate-CoA ligase (CNL) function as well as a reduction in the expression of the MYB transcription factor ODO1 explain the loss of scent during the transition from moth to hummingbird pollination. The CNL gene in the hummingbird-adapted species is inactive due to a stop codon, but also appears to have undergone further degradation over time. Therefore, we propose that loss of scent happened relatively early in the transition toward hummingbird pollination, and probably preceded the loss of UV-absorbing flavonols. The discovery that CNL is also involved in the loss of scent during the transition from outcrossing to selfing in Capsella (Brassicaceae) (see the accompanying paper) raises interesting questions about the possible causes of deep evolutionary conservation of the targets of evolutionary change.

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development.

Thermophilic growth and enzymatic thermostability are polyphyletic traits within Chaetomiaceae.

Thermophilic fungi have the potential to produce industrial-relevant thermostable enzymes, in particular for the degradation of plant biomass. Sordariales is one of the few fungal orders containing several thermophilic taxa, of which many have been associated with the production of thermostable enzymes. The evolutionary affiliation of Sordariales fungi, especially between thermophiles and non-thermophilic relatives, is however poorly understood. Phylogenetic analysis within the current study was based on sequence data, derived from a traditional Sanger and highly multiplexed targeted next generation sequencing approach of 45 isolates. The inferred phylogeny and detailed growth analysis rendered the trait 'thermophily' as polyphyletic within Chaetomiaceae (Sordariales, Sordariomycetes), and characteristic to: Myceliophthora spp., Thielavia terrestris, Chaetomium thermophilum, and Mycothermus thermophilus. Compared to mesophiles, the isolates within thermophilic taxa produced enzyme mixtures with the highest thermostability of known cellulase activities. Temperature profiles of the enzyme activities correlated strongly with the optimal growth temperatures of the isolates but not with their phylogenetic relationships. This strong correlation between growth and enzyme characteristics indicated that detailed analysis of growth does give predictive information on enzyme physiology. The variation in growth and enzyme characteristics reveals these fungi as an excellent platform to better understand fungal thermophily and enzyme thermostability.

Draft Genome Sequence of the Ant-Associated Fungus Phialophora attae (CBS 131958).

The black yeast Phialophora attae was isolated from the cuticle of tropical ant gynes. The ant-fungus association is sustained due to symbiotic evolutionary adaptations that allow fungal assimilation and tolerance of toxic compounds produced by the ant. The genome sequence of the first ant-associated fungus, P. attae, is presented here.

WRKY40 and WRKY6 act downstream of the green leaf volatile E-2-hexenal in Arabidopsis.

Plants are known to be responsive to volatiles, but knowledge about the molecular players involved in transducing their perception remains scarce. We study the response of Arabidopsis thaliana to E-2-hexenal, one of the green leaf volatiles (GLV) that is produced upon wounding, herbivory or infection with pathogens. We have taken a transcriptomics approach to identify genes that are induced by E-2-hexenal, but not by defence hormones or other GLVs. Furthermore, by studying the promoters of early E-2-hexenal-induced genes we determined that the only statistically enriched cis-element was the W-box motif. Since members of the plant-specific family of WRKY transcription factors act in trans on this cis-element, we focused on WRKY6, 40 and 53 that were most strongly induced by E-2-hexenal. Root elongation of Arabidopsis seedlings of the wrky40 wrky6 double mutant was much less inhibited than in wt plants, similar to the E-2-hexenal-responsive mutant her1, which is perturbed in γ-amino butyric acid (GABA) metabolism. The induction of several of the E-2-hexenal-specific genes was much higher in the wrky40, wrky6 or wrky40 wrky6 mutants, including GAD4, a glutamate decarboxylase that catalyzes the formation of GABA from glutamate. In conclusion, WRKY6 and 40 seem to act as important players transducing E-2-hexenal perception.

Viral metagenomics in drug-naïve, first-onset schizophrenia patients with prominent negative symptoms.

Although several studies suggest a virus or (endogenous) retrovirus involvement at the time of onset of schizophrenia, the unequivocal identification of one or more infectious agents, by means of an undirected catch-all technique, has never been conducted. In this study VIDISCA, a virus discovery method, was used in combination with Roche-454 high-throughput sequencing as a tool to determine the possible presence of viruses (known or unknown) in blood of first-onset drugs-naïve schizophrenic patients with prominent negative symptoms. Two viruses (the Anellovirus Torque Teno virus and GB virus C) were detected. Both viruses are commonly found in healthy individuals and no clear link with disease was ever established. Viruses from the family Anelloviridae were also identified in the control population (4.8%). Besides, one patient sample was positive for human endogenous retroviruses type K (HML-2) RNA but no specific predominant strain was detected, instead 119 different variants were found. In conclusion, these findings indicate no evidence for viral or endogenous retroviral involvement in sera at the time of onset of schizophrenia.

Whiteflies glycosylate salicylic acid and secrete the conjugate via their honeydew.

During insect feeding, a complex interaction takes place at the feeding site, with plants deciphering molecular information associated with the feeding herbivore, resulting in the upregulation of the appropriate defenses, and the herbivore avoiding or preventing these defenses from taking effect. Whiteflies can feed on plants without causing significant damage to mesophyll cells, making their detection extra challenging for the plant. However, whiteflies secrete honeydew that ends up on the plant surface at the feeding site and on distal plant parts below the feeding site. We reasoned that this honeydew, since it is largely of plant origin, may contain molecular information that alerts the plant, and we focused on the defense hormone salicylic acid (SA). First, we analyzed phloem sap from tomato plants, on which the whiteflies are feeding, and found that it contained salicylic acid (SA). Subsequently, we determined that in honeydew more than 80% of SA was converted to its glycoside (SAG). When whiteflies were allowed to feed from an artificial diet spiked with labeled SA, labeled SAG also was produced. However, manually depositing honeydew on undamaged plants resulted still in a significant increase in endogenous free SA. Accordingly, transcript levels of PR1a, an SA marker gene, increased whereas those of PI-II, a jasmonate marker gene, decreased. Our results indicate that whiteflies manipulate the SA levels within their secretions, thus influencing the defense responses in those plant parts that come into contact with honeydew.

Identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified VIDISCA.

BACKGROUND: The use of sequence independent methods combined with next generation sequencing for identification purposes in clinical samples appears promising and exciting results have been achieved to understand unexplained infections. One sequence independent method, Virus Discovery based on cDNA Amplified Fragment Length Polymorphism (VIDISCA) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures. METHODS: VIDISCA is normally combined with next generation sequencing, however, we set up a simplified VIDISCA which can be used in case next generation sequencing is not possible. Stool samples of 10 patients with unexplained acute flaccid paralysis showing cytopathic effect in rhabdomyosarcoma cells and/or mouse cells were used to test the efficiency of this method. To further characterize the viruses, VIDISCA-positive samples were amplified and sequenced with gene specific primers. RESULTS: Simplified VIDISCA detected seven viruses (70%) and the proportion of eukaryotic viral sequences from each sample ranged from 8.3 to 45.8%. Human enterovirus EV-B97, EV-B100, echovirus-9 and echovirus-21, human parechovirus type-3, human astrovirus probably a type-3/5 recombinant, and tetnovirus-1 were identified. Phylogenetic analysis based on the VP1 region demonstrated that the human enteroviruses are more divergent isolates circulating in the community. CONCLUSION: Our data support that a simplified VIDISCA protocol can efficiently identify unrecognized viruses grown in cell culture with low cost, limited time without need of advanced technical expertise. Also complex data interpretation is avoided thus the method can be used as a powerful diagnostic tool in limited resources. Redesigning the routine diagnostics might lead to additional detection of previously undiagnosed viruses in clinical samples of patients.

Metagenomic analysis of a sample from a patient with respiratory tract infection reveals the presence of a γ-papillomavirus.

Previously unknown or unexpected pathogens may be responsible for that proportion of respiratory diseases in which a causative agent cannot be identified. The application of broad-spectrum, sequence independent virus discovery techniques may be useful to reduce this proportion and widen our knowledge about respiratory pathogens. Thanks to the availability of high-throughput sequencing (HTS) technology, it became today possible to detect viruses which are present at a very low load, but the clinical relevance of those viruses must be investigated. In this study we used VIDISCA-454, a restriction enzyme based virus discovery method that utilizes Roche 454 HTS system, on a nasal swab collected from a subject with respiratory complaints. A γ-papillomavirus was detected (complete genome: 7142 bp) and its role in disease was investigated. Respiratory samples collected both during the acute phase of the illness and 2 weeks after full recovery contained the virus. The patient presented antibodies directed against the virus but there was no difference between IgG levels in blood samples collected during the acute phase and 2 weeks after full recovery. We therefore concluded that the detected γ-papillomavirus is unlikely to be the causative agent of the respiratory complaints and its presence in the nose of the patient is not related to the disease. Although HTS based virus discovery techniques proved their great potential as a tool to clarify the etiology of some infectious diseases, the obtained information must be subjected to cautious interpretations. This study underlines the crucial importance of performing careful investigations on viruses identified when applying sensitive virus discovery techniques, since the mere identification of a virus and its presence in a clinical sample are not satisfactory proofs to establish a causative link with a disease.

Unexplained diarrhoea in HIV-1 infected individuals.

BACKGROUND: Gastrointestinal symptoms, in particular diarrhoea, are common in non-treated HIV-1 infected individuals. Although various enteric pathogens have been implicated, the aetiology of diarrhoea remains unexplained in a large proportion of HIV-1 infected patients. Our aim is to identify the cause of diarrhoea for patients that remain negative in routine diagnostics. METHODS: In this study stool samples of 196 HIV-1 infected persons, including 29 persons with diarrhoea, were examined for enteropathogens and HIV-1. A search for unknown and unexpected viruses was performed using virus discovery cDNA-AFLP combined with Roche-454 sequencing (VIDISCA-454). RESULTS: HIV-1 RNA was detected in stool of 19 patients with diarrhoea (66%) compared to 75 patients (45%) without diarrhoea. In 19 of the 29 diarrhoea cases a known enteropathogen could be identified (66%). Next to these known causative agents, a range of recently identified viruses was identified via VIDISCA-454: cosavirus, Aichi virus, human gyrovirus, and non-A non-B hepatitis virus. Moreover, a novel virus was detected which was named immunodeficiency-associated stool virus (IASvirus). However, PCR based screening for these viruses showed that none of these novel viruses was associated with diarrhoea. Notably, among the 34% enteropathogen-negative cases, HIV-1 RNA shedding in stool was more frequently observed (80%) compared to enteropathogen-positive cases (47%), indicating that HIV-1 itself is the most likely candidate to be involved in diarrhoea. CONCLUSION: Unexplained diarrhoea in HIV-1 infected patients is probably not caused by recently described or previously unknown pathogens, but it is more likely that HIV-1 itself plays a role in intestinal mucosal abnormalities which leads to diarrhoea.

Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections.

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.

Performance of VIDISCA-454 in feces-suspensions and serum.

Virus discovery combining sequence unbiased amplification with next generation sequencing is now state-of-the-art. We have previously determined that the performance of the unbiased amplification technique which is operational at our institute, VIDISCA-454, is efficient when respiratory samples are used as input. The performance of the assay is, however, not known for other clinical materials like blood or stool samples. Here, we investigated the sensitivity of VIDISCA-454 with feces-suspensions and serum samples that are positive and that have been quantified for norovirus and human immunodeficiency virus type 1, respectively. The performance of VIDISCA-454 in serum samples was equal to its performance in respiratory material, with an estimated lower threshold of 1,000 viral genome copies. The estimated threshold in feces-suspension is around 200,000 viral genome copies. The decreased sensitivity in feces suspension is mainly due to sequences that share no recognizable identity with known sequences. Most likely these sequences originate from bacteria and phages which are not completely sequenced.

Evaluation of persistence of resistant variants with ultra-deep pyrosequencing in chronic hepatitis C patients treated with telaprevir.

BACKGROUND & AIMS: Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment. METHODS: Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103) were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline. RESULTS: Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing. CONCLUSION: In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare.