Evolutionary Relationships and Range Evolution of Greenhood Orchids (Subtribe Pterostylidinae): Insights From Plastid Phylogenomics.

Australia harbours a rich and highly endemic orchid flora with over 90% of native species found nowhere else. However, little is known about the assembly and evolution of Australia's orchid flora. Here, we used a phylogenomic approach to infer evolutionary relationships, divergence times and range evolution in Pterostylidinae (Orchidoideae), the second largest subtribe in the Australian orchid flora, comprising the genera Pterostylis and Achlydosa. Phylogenetic analysis of 75 plastid genes provided well-resolved and supported phylogenies. Intrageneric relationships in Pterostylis were clarified and monophyly of eight of 10 sections supported. Achlydosa was found to not form part of Pterostylidinae and instead merits recognition at subtribal level, as Achlydosinae. Pterostylidinae were inferred to have originated in eastern Australia in the early Oligocene, coinciding with the complete separation of Australia from Antarctica and the onset of the Antarctic Circumpolar Current, which led to profound changes in the world's climate. Divergence of all major lineages occurred during the Miocene, accompanied by increased aridification and seasonality of the Australian continent, resulting in strong vegetational changes from rainforest to more open sclerophyllous vegetation. The majority of extant species were inferred to have originated in the Quaternary, from the Pleistocene onwards. The rapid climatic oscillations during the Pleistocene may have acted as important driver of speciation in Pterostylidinae. The subtribe underwent lineage diversification mainly within its ancestral range, in eastern Australia. Long-distance dispersals to southwest Australia commenced from the late Miocene onwards, after the establishment of the Nullarbor Plain, which constitutes a strong edaphic barrier to mesic plants. Range expansions from the mesic into the arid zone of eastern Australia (Eremaean region) commenced from the early Pleistocene onwards. Extant distributions of Pterostylidinae in other Australasian regions, such as New Zealand and New Caledonia, are of more recent origin, resulting from long-distance dispersals from the Pliocene onwards. Temperate eastern Australia was identified as key source area for dispersals to other Australasian regions.

Assessing DNA for fish identifications from reference collections: the good, bad and ugly shed light on formalin fixation and sequencing approaches.

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.

Genome sequencing in persistently unsolved white matter disorders.

Genetic white matter disorders have heterogeneous etiologies and overlapping clinical presentations. We performed a study of the diagnostic efficacy of genome sequencing in 41 unsolved cases with prior exome sequencing, resolving an additional 14 from an historical cohort (n = 191). Reanalysis in the context of novel disease-associated genes and improved variant curation and annotation resolved 64% of cases. The remaining diagnoses were directly attributable to genome sequencing, including cases with small and large copy number variants (CNVs) and variants in deep intronic and technically difficult regions. Genome sequencing, in combination with other methodologies, achieved a diagnostic yield of 85% in this retrospective cohort.

Heterozygous Variants in the Mechanosensitive Ion Channel TMEM63A Result in Transient Hypomyelination during Infancy.

Mechanically activated (MA) ion channels convert physical forces into electrical signals. Despite the importance of this function, the involvement of mechanosensitive ion channels in human disease is poorly understood. Here we report heterozygous missense mutations in the gene encoding the MA ion channel TMEM63A that result in an infantile disorder resembling a hypomyelinating leukodystrophy. Four unrelated individuals presented with congenital nystagmus, motor delay, and deficient myelination on serial scans in infancy, prompting the diagnosis of Pelizaeus-Merzbacher (like) disease. Genomic sequencing revealed that all four individuals carry heterozygous missense variants in the pore-forming domain of TMEM63A. These variants were confirmed to have arisen de novo in three of the four individuals. While the physiological role of TMEM63A is incompletely understood, it is highly expressed in oligodendrocytes and it has recently been shown to be a MA ion channel. Using patch clamp electrophysiology, we demonstrated that each of the modeled variants result in strongly attenuated stretch-activated currents when expressed in naive cells. Unexpectedly, the clinical evolution of all four individuals has been surprisingly favorable, with substantial improvements in neurological signs and developmental progression. In the three individuals with follow-up scans after 4 years of age, the myelin deficit had almost completely resolved. Our results suggest a previously unappreciated role for mechanosensitive ion channels in myelin development.

Erratum: Author Correction: Pathogenic copy number variants that affect gene expression contribute to genomic burden in cerebral palsy.

[This corrects the article DOI: 10.1038/s41525-018-0073-4.].

Leukoencephalopathy due to variants in GFPT1-associated congenital myasthenic syndrome.

OBJECTIVE: To determine the molecular etiology of disease in 4 individuals from 2 unrelated families who presented with proximal muscle weakness and features suggestive of mitochondrial disease. METHODS: Clinical information and neuroimaging were reviewed. Genome sequencing was performed on affected individuals and biological parents. RESULTS: All affected individuals presented with muscle weakness and difficulty walking. In one family, both children had neonatal respiratory distress while the other family had 2 children with episodic deteriorations. In each family, muscle biopsy demonstrated ragged red fibers. MRI was suggestive of a mitochondrial leukoencephalopathy, with extensive deep cerebral white matter T2 hyperintense signal and selective involvement of the middle blade of the corpus callosum. Through genome sequencing, homozygous GFPT1 missense variants were identified in the affected individuals of each family. The variants detected (p.Arg14Leu and p.Thr151Lys) are absent from population databases and predicted to be damaging by in silico prediction tools. Following the genetic diagnosis, nerve conduction studies were performed and demonstrated a decremental response to repetitive nerve stimulation, confirming the diagnosis of myasthenia. Treatment with pyridostigmine was started in one family with favorable response. CONCLUSIONS: GFPT1 encodes a widely expressed protein that controls the flux of glucose into the hexosamine-biosynthesis pathway that produces precursors for glycosylation of proteins. GFPT1 variants and defects in other enzymes of this pathway have previously been associated with congenital myasthenia. These findings identify leukoencephalopathy as a previously unrecognized phenotype in GFPT1-related disease and suggest that mitochondrial dysfunction could contribute to this disorder.

Pathogenic copy number variants that affect gene expression contribute to genomic burden in cerebral palsy.

Cerebral palsy (CP) is the most frequent movement disorder of childhood affecting 1 in 500 live births in developed countries. We previously identified likely pathogenic de novo or inherited single nucleotide variants (SNV) in 14% (14/98) of trios by exome sequencing and a further 5% (9/182) from evidence of outlier gene expression using RNA sequencing. Here, we detected copy number variants (CNV) from exomes of 186 unrelated individuals with CP (including our original 98 trios) using the CoNIFER algorithm. CNV were validated with Illumina 850 K SNP arrays and compared with RNA-Seq outlier gene expression analysis from lymphoblastoid cell lines (LCL). Gene expression was highly correlated with gene dosage effect. We resolved an additional 3.7% (7/186) of this cohort with pathogenic or likely pathogenic CNV while a further 7.7% (14/186) had CNV of uncertain significance. We identified recurrent genomic rearrangements previously associated with CP due to 2p25.3 deletion, 22q11.2 deletions and duplications and Xp monosomy. We also discovered a deletion of a single gene, PDCD6IP, and performed additional zebrafish model studies to support its single allele loss in CP aetiology. Combined SNV and CNV analysis revealed pathogenic and likely pathogenic variants in 22.7% of unselected individuals with CP.

A recurrent de novo mutation in TMEM106B causes hypomyelinating leukodystrophy.

Hypomyelinating leukodystrophies are a heterogeneous group of disorders with a clinical presentation that often includes early-onset nystagmus, ataxia and spasticity and a wide range of severity. Using next-generation sequencing techniques and GeneMatcher, we identified four unrelated patients with brain hypomyelination, all with the same recurrent dominant mutation, c.754G>A p.(Asp252Asn), in TMEM106B. The mutation was confirmed as de novo in three of the cases, and the mildly affected father of the fourth affected individual was confirmed as mosaic for this variant. The protein encoded by TMEM106B is poorly characterized but is reported to have a role in regulation of lysosomal trafficking. Polymorphisms in TMEM106B are thought to modify disease onset in frontotemporal dementia, but its relation to myelination is not understood. Clinical presentation in three of the four patients is remarkably benign compared to other hypomyelinating disorders, with congenital nystagmus and mild motor delay. These findings add TMEM106B to the growing list of genes causing hypomyelinating disorders and emphasize the essential role lysosomes play in myelination.

X-linked hypomyelination with spondylometaphyseal dysplasia (H-SMD) associated with mutations in AIFM1.

An X-linked condition characterized by the combination of hypomyelinating leukodystrophy and spondylometaphyseal dysplasia (H-SMD) has been observed in only four families, with linkage to Xq25-27, and recent genetic characterization in two families with a common AIFM1 mutation. In our study, 12 patients (6 families) with H-SMD were identified and underwent comprehensive assessment accompanied by whole-exome sequencing (WES). Pedigree analysis in all families was consistent with X-linked recessive inheritance. Presentation typically occurred between 12 and 36 months. In addition to the two disease-defining features of spondylometaphyseal dysplasia and hypomyelination on MRI, common clinical signs and symptoms included motor deterioration, spasticity, tremor, ataxia, dysarthria, cognitive defects, pulmonary hypertension, nystagmus, and vision loss due to retinopathy. The course of the disease was slowly progressive. All patients had maternally inherited or de novo mutations in or near exon 7 of AIFM1, within a region of 70 bp, including synonymous and intronic changes. AIFM1 mutations have previously been associated with neurologic presentations as varied as intellectual disability, hearing loss, neuropathy, and striatal necrosis, while AIFM1 mutations in this small region present with a distinct phenotype implicating bone. Analysis of cell lines derived from four patients identified significant reductions in AIFM1 mRNA and protein levels in osteoblasts. We hypothesize that AIFM1 functions in bone metabolism and myelination and is responsible for the unique phenotype in this condition.

Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish.

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc-Vegfd-Ccbe1-Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.

Closely-related Borrelia burgdorferi (sensu stricto) strains exhibit similar fitness in single infections and asymmetric competition in multiple infections.

BACKGROUND: Wild hosts are commonly co-infected with complex, genetically diverse, pathogen communities. Competition is expected between genetically or ecologically similar pathogen strains which may influence patterns of coexistence. However, there is little data on how specific strains of these diverse pathogen species interact within the host and how this impacts pathogen persistence in nature. Ticks are the most common disease vector in temperate regions with Borrelia burgdorferi, the causative agent of Lyme disease, being the most common vector-borne pathogen in North America. Borrelia burgdorferi is a pathogen of high public health concern and there is significant variation in infection phenotype between strains, which influences predictions of pathogen dynamics and spread. METHODS: In a laboratory experiment, we investigated whether two closely-related strains of B. burgdorferi (sensu stricto) showed similar transmission phenotypes, how the transmission of these strains changed when a host was infected with one strain, re-infected with the same strain, or co-infected with two strains. Ixodes scapularis, the black-legged tick, nymphs were used to sequentially infect laboratory-bred Peromyscus leucopus, white-footed mice, with one strain only, homologous infection with the same stain, or heterologous infection with both strains. We used the results of this laboratory experiment to simulate long-term persistence and maintenance of each strain in a simple simulation model. RESULTS: Strain LG734 was more competitive than BL206, showing no difference in transmission between the heterologous infection groups and single-infection controls, while strain BL206 transmission was significantly reduced when strain LG734 infected first. The results of the model show that this asymmetry in competition could lead to extinction of strain BL206 unless there was a tick-to-host transmission advantage to this less competitive strain. CONCLUSIONS: This asymmetric competitive interaction suggests that strain identity and the biotic context of co-infection is important to predict strain dynamics and persistence.

Placental transcriptome co-expression analysis reveals conserved regulatory programs across gestation.

BACKGROUND: Mammalian development in utero is absolutely dependent on proper placental development, which is ultimately regulated by the placental genome. The regulation of the placental genome can be directly studied by exploring the underlying organisation of the placental transcriptome through a systematic analysis of gene-wise co-expression relationships. RESULTS: In this study, we performed a comprehensive analysis of human placental co-expression using RNA sequencing and intergrated multiple transcriptome datasets spanning human gestation. We identified modules of co-expressed genes that are preserved across human gestation, and also identifed modules conserved in the mouse indicating conserved molecular networks involved in placental development and gene expression patterns more specific to late gestation. Analysis of co-expressed gene flanking sequences indicated that conserved co-expression modules in the placenta are regulated by a core set of transcription factors, including ZNF423 and EBF1. Additionally, we identified a gene co-expression module enriched for genes implicated in the pregnancy pathology preeclampsia. By using an independnet transcriptome dataset, we show that these co-expressed genes are differentially expressed in preeclampsia. CONCLUSIONS: This study represents a comprehensive characterisation of placental co-expression and provides insight into potential transcriptional regulators that govern conserved molecular programs fundamental to placental development.

Effects of long-term tenofovir-based combination antiretroviral therapy in HIV-hepatitis B virus coinfection on persistent hepatitis B virus viremia and the role of hepatitis B virus quasispecies diversity.

OBJECTIVE: Hepatitis B virus (HBV) can persist in some HIV-HBV coinfected individuals on tenofovir disoproxil fumarate (TDF)-containing combination antiretroviral therapy (cART) but HBV resistance to TDF has not been reported and the source of persistent HBV DNA on TDF is poorly understood. The aims of this study were to assess long-term HBV suppression in HIV-HBV coinfected individuals receiving TDF and investigate quasispecies variation using ultradeep pyrosequencing (UDPS). METHODS: Ninety-two HIV-HBV coinfected participants on, or about to commence, TDF-containing cART were enrolled [Australia (n = 40), Thailand (n = 52)] and followed for 2 years with study visits every 6 months. HBV reverse transcriptase sequencing was performed on samples with HBV DNA more than 400 IU/ml by population-based methods and UDPS. Quasispecies diversity was assessed using Shannon entropy. RESULTS: Over 24 months, viremia was detected at least once in 17% (n = 16) of the cohort. Novel mutations were not identified in on TDF samples tested by population-based sequencing (n = 19). Using UDPS, the median Shannon entropy value in samples prior to TDF in patients aviremic on TDF was not statistically different from those who were viremic on TDF (n = 50; 8.4 and 9.1, respectively, P = 0.9). Longitudinal Shannon entropy analysis of on TDF samples from five participants showed three individuals with significant changes in viral diversity over time. CONCLUSION: Persistent viremia on TDF-containing cART is common but TDF-resistance was not detected. In some individuals, changes in viral diversity over time were observed on TDF which could potentially be active viral replication. Further follow-up will be needed to determine the clinical significance of detectable HBV DNA on TDF-containing cART.

Genomic Comparison of Two O111:H- Enterohemorrhagic Escherichia coli Isolates from a Historic Hemolytic-Uremic Syndrome Outbreak in Australia.

Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhea and hemolytic-uremic syndrome (HUS) worldwide. Australia's worst outbreak of HUS occurred in Adelaide in 1995 and was one of the first major HUS outbreaks attributed to a non-O157 Shiga-toxigenic E. coli (STEC) strain. Molecular analyses conducted at the time suggested that the outbreak was caused by an O111:H(-) clone, with strains from later in the outbreak harboring an extra copy of the genes encoding the potent Shiga toxin 2 (Stx2). Two decades later, we have used next-generation sequencing to compare two isolates from early and late in this important outbreak. We analyzed genetic content, single-nucleotide polymorphisms (SNPs), and prophage insertion sites; for the latter, we demonstrate how paired-end sequence data can be leveraged to identify such insertion sites. The two strains are genetically identical except for six SNP differences and the presence of not one but two additional Stx2-converting prophages in the later isolate. Isolates from later in the outbreak were associated with higher levels of morbidity, suggesting that the presence of the additional Stx2-converting prophages is significant in terms of the virulence of this clone.

A discrete role for FNR in the transcriptional response to moderate changes in oxygen by Haemophilus influenzae Rd KW20.

The survival by pathogenic bacteria within the specific conditions of an anatomical niche is critical for their persistence. These conditions include the combination of toxic chemicals, such as reactive oxygen (ROS) and reactive nitrogen species (RNS), with factors relevant to cell growth, such as oxygen. Haemophilus influenzae senses oxygen levels largely through the redox state of the intracellular fumarate-nitrate global regulator (FNR). H. influenzae certainly encounters oxygen levels that fluctuate, but in reality, these would rarely reach a state that results in FNR being fully reduced or oxidized. We were therefore interested in the response of H. influenzae to ROS and RNS at moderately high or low oxygen levels and the corresponding role of FNR. At these levels of oxygen, even though the growth rate of an H. influenzae fnr mutant was similar to wild type, its ROS and RNS tolerance was significantly different. Additionally, the subtle changes in oxygen did alter the whole cell transcriptional profile and this was different between the wild type and fnr mutant strains. It was the changed whole cell profile that impacted on ROS/RNS defence, but surprisingly, the FNR-regulated, anaerobic nitrite reductase (NrfA) continued to be expressed and had a role in this phenotype.

Borrelia burgdorferi sensu stricto and Borrelia afzelii: Population structure and differential pathogenicity.

MultiLocus sequence typing (MLST) is considered a powerful method to unveil relationships within bacterial populations and it constitutes an economical and fast alternative to whole genome sequencing. We used this method to understand whether there are differences in human pathogenicity within and between different Borrelia burgdorferi sensu lato species. Therefore, 136 strains from human patients or ticks from Europe were included in MLST analyses. The scheme employed used eight chromosomally located housekeeping genes (i.e. clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA). We investigated Borrelia afzelii, one of the predominant species in Europe, and B. burgdorferi sensu stricto (s.s.), because it allowed comparative analysis to strains from the USA. We typed 113 patient isolates as well as 23 tick isolates. For further comparative purposes an additional 746 strains from Europe and the USA were included from the MLST website http://borrelia.mlst.net. We observed an overlap of the B. burgdorferi s.s. populations from Europe and the USA isolated from human patients while there was no overlap of the populations found in tick vectors. Further results indicate that B. afzelii was significantly less associated with disseminated infection than B. burgdorferi s.s. and that B. burgdorferi s.s. from Europe caused neuroborreliosis to a significantly greater extent than B. afzelii or B. burgdorferi s.s. in the USA. Our data suggest that there may be an evolutionary basis of differential interspecies pathogenicity in Borrelia. This was not evident within Borrelia species: we found the same sequence types in patients with disseminated or localized symptoms when the number of strains was sufficiently high. We hypothesize that the finding that B. burgdorferi s.s. in Europe is much more associated with neuroborreliosis than in the USA maybe linked to factor(s) related to the human host, the tick vector or the bacterium itself (e.g. plasmid content and structure).

Whole genome capture of vector-borne pathogens from mixed DNA samples: a case study of Borrelia burgdorferi.

BACKGROUND: Rapid and accurate retrieval of whole genome sequences of human pathogens from disease vectors or animal reservoirs will enable fine-resolution studies of pathogen epidemiological and evolutionary dynamics. However, next generation sequencing technologies have not yet been fully harnessed for the study of vector-borne and zoonotic pathogens, due to the difficulty of obtaining high-quality pathogen sequence data directly from field specimens with a high ratio of host to pathogen DNA. RESULTS: We addressed this challenge by using custom probes for multiplexed hybrid capture to enrich for and sequence 30 Borrelia burgdorferi genomes from field samples of its arthropod vector. Hybrid capture enabled sequencing of nearly the complete genome (~99.5 %) of the Borrelia burgdorferi pathogen with 132-fold coverage, and identification of up to 12,291 single nucleotide polymorphisms per genome. CONCLUSIONS: The proprosed culture-independent method enables efficient whole genome capture and sequencing of pathogens directly from arthropod vectors, thus making population genomic study of vector-borne and zoonotic infectious diseases economically feasible and scalable. Furthermore, given the similarities of invertebrate field specimens to other mixed DNA templates characterized by a high ratio of host to pathogen DNA, we discuss the potential applicabilty of hybrid capture for genomic study across diverse study systems.

A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species.

Inducible and reversible lentiviral and Recombination Mediated Cassette Exchange (RMCE) systems for controlling gene expression.

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

Generation of a chimeric hepatitis C replicon encoding a genotype-6a NS3 protease and assessment of boceprevir (SCH503034) sensitivity and drug-associated mutations.

BACKGROUND: Genotype (gt)6 HCV is common amongst HCV-positive populations of the Asia-Pacific region but cell culture models for this gt have only recently been developed. Boceprevir (SCH503034) is a clinically available inhibitor of the HCV NS3 protein. We investigated the efficacy of boceprevir for inhibiting replication of a chimeric gt1b replicon encoding a gt6a NS3 protease and defined the development of mutations in the protease when boceprevir treatment was applied. METHODS: We constructed a chimeric gt1b subgenomic replicon encoding a gt6 NS3 protease (NS3p) sequence (gt6NS3p_gt1b). The boceprevir EC50 value against replication of this replicon was determined using quantitative reverse transcriptase PCR. Next-generation sequencing was used to identify nucleotide changes associated with boceprevir resistance. The replication capacities of chimeric replicons containing mutations associated with boceprevir resistance were determined by colony formation efficiency assays. RESULTS: The boceprevir EC50 value for the gt6NS3p_gt1b replicon was 535 ±79 nM. Boceprevir-resistant gt6NS3p_gt1b replicon cell lines could be selected and they demonstrated drug-associated amino acid changes that have previously been reported in other HCV gts. Interestingly, no mutations were observed at A156, a position defined for boceprevir resistance in gt1 NS3p, while mutation at N122, which is rarely reported in boceprevir-resistant gt1 proteases, was frequently observed. Re-introduction of these mutations into the chimeric replicon altered their replication capacity, ranging from complete abolishment of replication (A156T) to increasing replication capacity (V36A, N122S). This report provides the first characterization of gt6 HCV resistance to boceprevir. CONCLUSIONS: A chimeric HCV replicon encoding gt6 NS3 protease is sensitive to boceprevir and develops drug-resistant mutations at amino acid sites previously reported for other gts. Mutation at N122 also appears to be associated with boceprevir resistance in the gt6 NS3 protease.