Cost-utility analysis of sacituzumab govitecan versus chemotherapy for the treatment of metastatic triple-negative breast cancer in Singapore.

OBJECTIVE: To assess the cost-effectiveness of sacituzumab govitecan for treating relapsed or refractory metastatic triple-negative breast cancer (TNBC) in Singapore. METHODS: A three-state partitioned survival model was developed to evaluate the cost-effectiveness of sacituzumab govitecan from a healthcare system perspective over 5 years. Clinical inputs were obtained from the ASCENT trial. Health state utilities were retrieved from the literature and direct costs were sourced from public healthcare institutions in Singapore. Sensitivity and scenario analyses were conducted to explore the impact of uncertainties and assumptions on cost-effectiveness results. RESULTS: Compared with single-agent chemotherapy, sacituzumab govitecan was associated with a base-case incremental cost-effectiveness ratio (ICER) of S$328,000 (US$237,816) per quality-adjusted life year (QALY) gained. One-way sensitivity analyses showed that the ICER was most sensitive to the cost of sacituzumab govitecan and progression-free utility values. Regardless of variation in these parameters, the ICER remained high, and a substantial price reduction was required to reduce the ICER. CONCLUSION: At its current price, sacituzumab govitecan does not represent a cost-effective treatment for relapsed or refractory metastatic TNBC in Singapore. Our findings will be useful to inform funding decisions alongside other factors including clinical effectiveness, safety, and budget impact considerations.

Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation.

The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry.

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.

Inhibition of Calcium Dependent Protein Kinase 1 (CDPK1) by Pyrazolopyrimidine Analogs Decreases Establishment and Reoccurrence of Central Nervous System Disease by Toxoplasma gondii.

Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are critical to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goal of increasing selectivity over host enzymes, improving antiparasite potency, and improving metabolic stability. The resulting lead compound 24 exhibited excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, and decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.

A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission.

To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.Protein kinases are promising drug targets for treatment of malaria. Here, starting with a medicinal chemistry approach, Baker et al. generate an imidazopyridine that selectively targets Plasmodium falciparum PKG, inhibits blood stage parasite growth in vitro and in mice and blocks transmission to mosquitoes.

Discovery and structure activity relationship of the first potent cryptosporidium FIKK kinase inhibitor.

FIKKs are parasite-specific protein kinases with distinctive sequence motifs and their biological roles have not been completely elucidated. Here, we report the first potent Cryptosporidium FIKK (CpFIKK) inhibitor. We identified 4b as a potent (IC50=0.2nM) inhibitor of CpFIKK catalytic activity. In addition, we identified both CpCDPK1 selective as well as dually acting CpFIKK-CDPK1 inhibitors from the same structural class of compounds. We evaluated these CpFIKK inhibitors for inhibition of parasite growth in vitro. The observed effects on parasite growth did not correlate with CpFIKK inhibition, suggesting that CpFIKK may not be involved in parasite growth.

Discovery of a PCAF Bromodomain Chemical Probe.

The p300/CBP-associated factor (PCAF) and related GCN5 bromodomain-containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine-based L-45 (dubbed L-Moses) as the first potent, selective, and cell-active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)-(-)-norephedrine furnished L-45 in enantiopure form. L-45 was shown to disrupt PCAF-Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co-crystal structure of L-45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L-45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell-permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Identification of small molecule inhibitors that block the Toxoplasma gondii rhoptry kinase ROP18.

The protozoan parasite Toxoplasma gondii secretes a family of serine-threonine protein kinases into its host cell in order to disrupt signaling and alter immune responses. One prominent secretory effector is the rhoptry protein 18 (ROP18), a serine-threonine kinase that phosphorylates immunity related GTPases (IRGs) and hence blocks interferon gamma-mediated responses in rodent cells. Previous genetic studies show that ROP18 is a major virulence component of T. gondii strains from North and South America. Here, we implemented a high throughput screen to identify small molecule inhibitors of ROP18 in vitro and subsequently validated their specificity within infected cells. Although ROP18 was not susceptible to many kinase-directed inhibitors that affect mammalian kinases, the screen identified several sub micromolar inhibitors that belong to three chemical scaffolds: oxindoles, 6-azaquinazolines, and pyrazolopyridines. Treatment of interferon gamma-activated cells with one of these inhibitors enhanced immunity related GTPase recruitment to wild type parasites, recapitulating the defect of Δrop18 mutant parasites, consistent with targeting ROP18 within infected cells. These compounds provide useful starting points for chemical biology experiments or as leads for therapeutic interventions designed to reduce parasite virulence.

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling.

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca(2+) Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca(2+) indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca(2+) signaling in the model apicomplexan Toxoplasma gondii In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca(2+) We define the pool of Ca(2+) regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca(2+) signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca(2+) The enhancers identified are capable of releasing intracellular Ca(2+) stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum Inhibition of Ca(2+)-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca(2+) stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca(2+), underscoring the importance of these pathways and the therapeutic potential of their inhibition.

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds.

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds' mechanisms of action--i.e., the specific molecular targets by which they kill the parasite--would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children's Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 µM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.

Topical imiquimod before intradermal trivalent influenza vaccine for protection against heterologous non-vaccine and antigenically drifted viruses: a single-centre, double-blind, randomised, controlled phase 2b/3 trial.

BACKGROUND: Pretreatment with topical imiquimod, a synthetic agonist of toll-like receptor 7, significantly improved the immunogenicity of influenza vaccination in elderly people. We aimed to clarify its effect in a younger age group. METHODS: In this double-blind, randomised controlled trial, we enrolled healthy volunteers aged 18-30 years in early 2014 to receive the 2013-14 northern-hemisphere winter trivalent influenza vaccine at the Queen Mary Hospital, (Hong Kong, China). Eligible participants were randomly assigned (1:1:1:1) to one of the four vaccination groups: the study group, topical imiquimod-cream followed by intradermal trivalent influenza vaccine (INF-Q-ID), or one of three control groups, topical aqueous-cream control followed by intradermal trivalent influenza vaccine (INF-C-ID), topical aqueous-cream control followed by intramuscular trivalent influenza vaccine (INF-C-IM), and topical imiquimod-cream followed by intradermal normal-saline injection (SAL-Q-ID). Randomisation was by computer-generated lists in blocks of four. The type of topical treatment was masked from volunteers and investigators, although not from the study nurse. Serum haemagglutination-inhibition and microneutralisation-antibody titres were assayed. The primary outcome was seroconversion at day 7 after treatment for three vaccine strains of influenza (A/California/07/2009 H1N1-like virus [A/California/H1N1], A/Victoria/361/2011 H3N2-like virus [A/Victoria/H3N2], and B/Massachusetts/2/2012-like virus [B/Yamagata lineage]) and four non-vaccine strains (A/HK/485197/14 [H3N2 Switzerland-like lineage], prototype A/WSN/1933 [H1N1], A/HK/408027/09 [prepandemic seasonal H1N1], and B/HK/418078/11 [Victoria lineage]). Analysis was done on an intention-to-treat basis. This trial is registered with ClinicalTrials.gov, number NCT02103023. FINDINGS: We enrolled 160 healthy volunteers between March 1 and May 31, 2014, and 40 participants were randomly assigned to each study group. For the A/California/H1N1 strain, seroconversion at day 7 occurred in 39 participants (98%) in the INF-Q-ID group, 25 (63%) in the INF-C-ID group, 18 (45%) in the INF-C-IM group, and none in the SAL-Q-ID group; for the A/Victoria/H3N2, this was 30 (75%) in the INF-Q-ID group, four (10%) in the INF-C-ID group, four (10%) in the INF-C-IM group, and none in the SAL-Q-ID group; and for the B/Massachusetts (Yamagata lineage) strain, this was 36 (90%) in the INF-Q-ID group, 27 (68%) in the INF-C-ID group, 17 (43%) in the INF-C-IM group, and one (3%) in the SAL-Q-ID group (p<0·0001 for all three vaccine strains). Adverse reactions were infrequent and self-limited and did not differ between the four groups. Furthermore, the seroconversion rate against the four non-vaccine strains was better in the INF-Q-ID group than in the control groups on days 7 and 21 (p<0·0001). The most common adverse events were grade 1 redness (five participants in the INF-Q-ID group, three in INF-C-ID, one in INF-C-IM, and one in SAL-Q-ID) and grade 1 swelling (seven participants in INF-Q-ID group, five in INF-C-ID, three in INF-C-IM, and two in SAL-Q-ID. INTERPRETATION: Topical application of imiquimod before intradermal trivalent influenza vaccine significantly improved immunogenicity against the vaccine influenza strains in young healthy individuals and increased immunogenicity against the non-vaccine strains, especially the antigenically drifted H3N2 strain of 2015, which was not included in the 2013-14 recommended vaccine. Further studies should be done to establish the efficacy and safety of this approach for other injectable vaccines to augment the onset and range of protection. FUNDING: The Shaw Foundation Hong Kong, Health and Medical Research Fund (Hong Kong, China), The Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the HKSAR (Department of Health, Hong Kong, China), The Providence Foundation, Respiratory Viral Research Foundation.

Biochemical characterization of FIKK8--A unique protein kinase from the malaria parasite Plasmodium falciparum and other apicomplexans.

FIKKs are protein kinases with distinctive sequence motifs found exclusively in Apicomplexa. Here, we report on the biochemical characterization of Plasmodium falciparum FIKK8 (PfFIKK8) and its Cryptosporidium parvum orthologue (CpFIKK) - the only member of the family predicted to be cytosolic and conserved amongst non-Plasmodium parasites. Recombinant protein samples of both were catalytically active. We characterized their phosphorylation ability using an enzymatic assay and substrate specificities using an arrayed positional scanning peptide library. Our results show that FIKK8 targets serine, preferably with arginine in the +3 and -3 positions. Furthermore, the soluble and active FIKK constructs in our experiments contained an N-terminal extension (NTE) conserved in FIKK8 orthologues from other apicomplexan species. Based on our results, we propose that this NTE is an integral feature of the FIKK subfamily.

Differential Expression Profile of Chicken Embryo Fibroblast DF-1 Cells Infected with Cell-Adapted Infectious Bursal Disease Virus.

RNA-Seq was used to unveil the transcriptional profile of DF-1 cells at the early stage of caIBDV infection. Total RNAs were extracted from virus-infected cells at 0, 6 and 12 hpi. RNA-Seq datasets of respective samples mapped to 56.5-57.6% of isoforms in the reference genome Galgal4.73. At 6 hpi, 23 isoforms underwent an elevated expression, while 128 isoforms were up-regulated and 5 were down-regulated at 12 hpi in the virus-infected group. Besides, 10 isoforms were exclusively expressed in the virus-infected cells. Though no significant change was detected in cytokine and interferon expression levels at the first 12 hours of infection, modulations of the upstream regulators were observed. In addition to the reported regulatory factors including EIF2AK2, MX, OAS*A, GBP7 and IFIT, IBDV infection also triggered a IFIT5-IRF1/3-RSAD5 pathway in the DF-1 cells which potentially restricted the viral replication cycle in the early infection stage. Over-expression of LIPA and CH25H, together with the suppression of STARD4, LSS and AACS genes implied a modulation of membrane fluidity and lipid raft arrangement in the infected cells. Alternative splicing of the EFR3 homolog A gene was also through to be involved in the lipid membrane regulation, and these cumulative responses projected an inhibition of viral endocytosis. Recognition of viral RNA genomes and intermediates was presumably enhanced by the elevated levels of IFIH1, DHX58 and TRIM25 genes which possess properties on detecting viral dsRNA. On the other hand, the caIBDV arrested the host's apoptotic process by inducing the expression of apoptosis inhibitors including NFKBIA/Z, TNFAIP2/3 and ITA at the first 12 hours of infection. In conclusion, the differential expression landscape demonstrated with RNA-Seq provides a comprehensive picture on the molecular interactions between host cells and virus at the early stage of infection.

Designing selective inhibitors for calcium-dependent protein kinases in apicomplexans.

Apicomplexan parasites cause some of the most severe human diseases, including malaria (caused by Plasmodium), toxoplasmosis, and cryptosporidiosis. Treatments are limited by the lack of effective drugs and development of resistance to available agents. By exploiting novel features of protein kinases in these parasites, it may be possible to develop new treatments. We summarize here recent advances in identifying small molecule inhibitors against a novel family of plant-like, calcium-dependent kinases that are uniquely expanded in apicomplexan parasites. Analysis of the 3D structure, activation mechanism, and sensitivity to small molecules had identified several attractive chemical scaffolds that are potent and selective inhibitors of these parasite kinases. Further optimization of these leads may yield promising new drugs for treatment of these parasitic infections.

Solution-state NMR structure of the putative morphogene protein BolA (PFE0790c) from Plasmodium falciparum.

Protozoa of the genus Plasmodium are responsible for malaria, which is perhaps the most important parasitic disease to infect mankind. The emergence of Plasmodium strains resistant to current therapeutics and prophylactics makes the development of new treatment strategies urgent. Among the potential targets for new antimalarial drugs is the BolA-like protein PFE0790c from Plasmodium falciparum (Pf-BolA). While the function of BolA is unknown, it has been linked to cell morphology by regulating transcription in response to stress. Using an NMR-based method, an ensemble of 20 structures of Pf-BolA was determined and deposited in the PDB (PDB entry 2kdn). The overall topology of the Pf-BolA structure, α1-ß1-ß2-η1-α2/η2-ß3-α3, with the ß-strands forming a mixed ß-sheet, is similar to the fold observed in other BolA structures. A helix-turn-helix motif similar to the class II KH fold associated with nucleic acid-binding proteins is present, but contains an FXGXXXL signature sequence that differs from the GXXG signature sequence present in class II KH folds, suggesting that the BolA family of proteins may use a novel protein-nucleic acid interface. A well conserved arginine residue, Arg50, hypothesized to play a role in governing the formation of the C-terminal α-helix in the BolA family of proteins, is too distant to form polar contacts with any side chains in this α-helix in Pf-BolA, suggesting that this conserved arginine may only serve a role in guiding the orientation of this C-terminal helix in some BolA proteins. A survey of BolA structures suggests that the C-terminal helix may not have a functional role and that the third helix (α2/η2) has a `kink' that appears to be conserved among the BolA protein structures. Circular dichroism spectroscopy shows that Pf-BolA is fairly robust, partially unfolding when heated to 353 K and refolding upon cooling to 298 K.

Structural and evolutionary divergence of cyclic nucleotide binding domains in eukaryotic pathogens: Implications for drug design.

Many cellular functions in eukaryotic pathogens are mediated by the cyclic nucleotide binding (CNB) domain, which senses second messengers such as cyclic AMP and cyclic GMP. Although CNB domain-containing proteins have been identified in many pathogenic organisms, an incomplete understanding of how CNB domains in pathogens differ from other eukaryotic hosts has hindered the development of selective inhibitors for CNB domains associated with infectious diseases. Here, we identify and classify CNB domain-containing proteins in eukaryotic genomes to understand the evolutionary basis for CNB domain functional divergence in pathogens. We identify 359 CNB domain-containing proteins in 31 pathogenic organisms and classify them into distinct subfamilies based on sequence similarity within the CNB domain as well as functional domains associated with the CNB domain. Our study reveals novel subfamilies with pathogen-specific variations in the phosphate-binding cassette. Analyzing these variations in light of existing structural and functional data provides new insights into ligand specificity and promiscuity and clues for drug design. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Complete Genome Sequence of Dyella japonica Strain A8 Isolated from Malaysian Tropical Soil.

We previously identified and presented the draft genome of a Xanthomonadaceae bacterial strain Dyella japonica A8 which shows quorum-quenching activity. Here, we report the complete, closed genome sequence of this bacterium. This complete genome may help to further investigate the comparative quorum-quenching activity among D. japonica strains.

Genomic evolution of porcine reproductive and respiratory syndrome virus (PRRSV) isolates revealed by deep sequencing.

Most studies on PRRSV evolution have been limited to a particular region of the viral genome. A thorough genome-wide understanding of the impact of different mechanisms on shaping PRRSV genetic diversity is still lacking. To this end, deep sequencing was used to obtain genomic sequences of a diverse set of 16 isolates from a region of Hong Kong with a complex PRRSV epidemiological record. Genome assemblies and phylogenetic typing indicated the co-circulation of strains of both genotypes (type 1 and type 2) with varying Nsp2 deletion patterns and distinct evolutionary lineages ("High Fever"-like and local endemic type). Recombination analyses revealed genomic breakpoints in structural and non-structural regions of genomes of both genotypes with evidence of many recombination events originating from common ancestors. Additionally, the high fold of coverage per nucleotide allowed the characterization of minor variants arising from the quasispecies of each strain. Overall, 0.56-2.83% of sites were found to be polymorphic with respect to cognate consensus genomes. The distribution of minor variants across each genome was not uniform indicating the influence of selective forces. Proportion of variants capable of causing an amino acid change in their respective codons ranged between 25-67% with many predicted to be non-deleterious. Low frequency deletion variants were also detected providing one possible mechanism for their sudden emergence as cited in previous reports.