Genomic Clones Research Articles

Targeted knockout of <italic>Kcna3</italic> and its function in Jurkat T cells using the CRISPR/Cas9 system

To explore the use of the CRISPR/Cas9 system to target Kv1.3 channel-encoding genes Kcna3 and elucidate the pathophysiological function mediated by Kv1.3 channels in Jurkat T cells. The first exon sgRNA of human Kcna3 in pX458-sgRNA recombinant vector was transferred into human Jurkat T cells via electroporation. T7EN1 digestion, Genomic PCR products and TA cloning sequencing, Western blot, and functional assays, such as electrophysiology, live-cell calcium imaging and ELISA were used to validate Kcna3 knockout effect in Jurkat T cells. The results of the sequencing analysis showed that the CRISPR/Cas9 recombinant plasmids pX458-sgRNA targeting Kcna3 were successfully constructed. The designed sgRNA2 can efficiently cleave genomic DNA. Kv1.3 protein expression could not be detected using Western blot analysis. Genomic PCR products and TA cloning sequencing showed the presence of Indel mutation. Whole-cell patch clamp experiments showed that Kv1.3 channel currents could not be recorded in Kcna3 knockout Jurkat T cells. Live-cell calcium imaging showed that the increase in free Ca2+ concentration in Kcna3 knockout Jurkat T cells was significantly lower than that in wild type Jurkat T cells. ELISA results showed that the IL-2 level of Kcna3 knockout Jurkat T cells was significantly decreased compared with that in wild-type Jurkat T cells ( P Kcna3 in Jurkat T cells using the CRISPR/Cas9 system strongly proved that Kv1.3 mediated the immune inflammatory response of Jurkat T cells, which provides a novel cell model for further study of the pathophysiological function of the Kv1.3 channel.

In vitro selection of aztreonam/avibactam resistance in dual-carbapenemase-producing Klebsiella pneumoniae.

To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.

Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

CReasPy-Cloning: A Method for Simultaneous Cloning and Engineering of Megabase-Sized Genomes in Yeast Using the CRISPR-Cas9 System.

Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.

The Role of Rice Vacuolar Invertase2 in Seed Size Control.

Sink strength optimizes sucrose import, which is fundamental to support developing seed grains and increase crop yields, including those of rice (Oryza sativa). In this regard, little is known about the function of vacuolar invertase (VIN) in controlling sink strength and thereby seed size. Here, in rice we analyzed mutants of two VINs, OsVIN1 and OsVIN2, to examine their role during seed development. In a phenotypic analysis of the T-DNA insertion mutants, only the OsVIN2 mutant osvin2-1 exhibited reduced seed size and grain weight. Scanning electron microscopy analysis revealed that the small seed grains of osvin2-1 can be attributed to a reduction in spikelet size. A significant decrease in VIN activity and hexose level in the osvin2-1 spikelets interfered with spikelet growth. In addition, significant reduction in starch and increase in sucrose, which are characteristic features of reduced turnover and flux of sucrose due to impaired sink strength, were evident in the pre-storage stage of osvin2-1 developing grains. In situ hybridization analysis found that expression of OsVIN2 was predominant in the endocarp of developing grains. A genetically complemented line with a native genomic clone of OsVIN2 rescued reduced VIN activity and seed size. Two additional mutants, osvin2-2 and osvin2-3 generated by the CRISPR/Cas9 method, exhibited phenotypes similar to those of osvin2-1 in spikelet and seed size, VIN activity, and sugar metabolites. These results clearly demonstrate an important role of OsVIN2 as sink strength modulator that is critical for the maintenance of sucrose flux into developing seed grains.

Glycine receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the zinc activated channels, GABAA, nicotinic acetylcholine and 5-HT3 receptors [63]. The receptor is expressed either as a homo-pentamer of α subunits, or a complex now thought to harbour 2α and 3β subunits [30, 7], that contain an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1INS and α1del), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [80, 91, 18]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the mature form of the receptor contains α1 (or α3) and β subunits while the immature form is mostly composed of only α2 subunits. RNA transcripts encoding the α4-subunit have not been detected in adult humans. The N-terminal domain of the α-subunit contains both the agonist and strychnine binding sites that consist of several discontinuous regions of amino acids. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. The latter is a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [86, 51, 53]. G-protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [122, 121]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [94]. Intracellular Ca2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [24].

Allometric Relationships for Aboveground Woody Biomass Differ Among Hybrid Poplar Genomic Groups and Clones in the North-Central USA

Allometric biomass equations were developed based on harvests of 198 trees from 15 field sites in the north-central USA, with the trees representing 4 hybrid poplar genomic groups and a total of 11 clones within these groups. Specifically, equations were developed to describe woody (branch + stem) total dry weight (TDW) as a function of diameter at breast height (DBH), along with hypothesis tests of differences among genomic groups and clones for equation intercepts and slopes. Inclusion of groups or clones improved model fit (r2 = 0.90 or 0.91, respectively) compared to the generic model consisting of only DBH (r2 = 0.85). Differences in equation parameters translated into significant differences among groups and clones for estimated TDW when compared at mean DBH (20 cm). Equations were also developed to describe branch-to-stem weight ratio (BSR) as a function of TDW and tree height (H), also with hypothesis tests of differences in intercepts and slopes among genomic groups and clones. Inclusion of genomic groups somewhat improved model fit (r2 = 0.57) compared to the generic model consisting of only TDW and H (r2 = 0.53), whereas model fit improved more markedly with the inclusion of clones (r2 = 0.75). Our results indicate that group- and clone-specific equations (rather than generic ones) are warranted for hybrid poplars, and that group-specific equations are adequate for estimating TDW whereas clone-specific equations are more appropriate for estimating BSR.

Cloning and characterisation of the mouse fra-2 gene.

Transcription factor AP-1 is comprised of multiple protein complexes that include members of a family of genes related to the proto-oncogene c-fos. In this report, we have extended the analysis of one member of this family, fos-related antigen-2 (fra-2), by isolating and characterising genomic and cDNA clones encoding the mouse fra-2 homolog. The overall gene structure (number and positions of introns) was similar to that of both the chicken fra-2 gene and other members of the fos family, and the relative positions of putative enhancers in the 5' regulatory region were well conserved between the mouse and chicken fra-2 genes. High levels of fra-2 mRNA were detected in ovary, stomach, small and large intestine, brain, lung and heart. The mouse Fra-2 protein showed 94% and 87.5% conservation with human and chicken Fra-2, respectively, and mouse Fra-2, like the chicken homolog, induced transformation of chicken embryo fibroblasts. The characterisation of the mouse fra-2 gene provides a basis for analysis of Fra-2 function in the whole animal.

Plant Virus Vectors 3.0: Transitioning into Synthetic Genomics.

Plant viruses were first implemented as heterologous gene expression vectors more than three decades ago. Since then, the methodology for their use has varied, but we propose it was the merging of technologies with virology tools, which occurred in three defined steps discussed here, that has driven viral vector applications to date. The first was the advent of molecular biology and reverse genetics, which enabled the cloning and manipulation of viral genomes to express genes of interest (vectors 1.0). The second stems from the discovery of RNA silencing and the development of high-throughput sequencing technologies that allowed the convenient and widespread use of virus-induced gene silencing (vectors 2.0). Here, we briefly review the events that led to these applications, but this treatise mainly concentrates on the emerging versatility of gene-editing tools, which has enabled the emergence of virus-delivered genetic queries for functional genomics and virology (vectors 3.0).

Seeking active RubisCOs from the currently uncultured microbial majority colonizing deep-sea hydrothermal vent environments.

Almost all the inorganic carbon on Earth is converted into biomass via the Calvin-Benson-Bassham (CBB) cycle. Here, the central carboxylation reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which can be found in numerous primary producers including plants, algae, cyanobacteria, and many autotrophic bacteria. Although RubisCO possesses a crucial role in global biomass production, it is not a perfect catalyst. Therefore, research interest persists on accessing the full potential of yet unexplored RubisCOs. We recently developed an activity-based screen suited to seek active recombinant RubisCOs from the environment-independent of the native host's culturability. Here, we applied this screen to twenty pre-selected genomic fosmid clones from six cultured proteobacteria to demonstrate that a broad range of phylogenetically distinct RubisCOs can be targeted. We then screened 12,500 metagenomic fosmid clones from six distinct hydrothermal vents and identified forty active RubisCOs. Additional sequence-based screening uncovered eight further RubisCOs, which could then also be detected by a modified version of the screen. Seven were active form III RubisCOs from yet uncultured Archaea. This indicates the potential of the activity-based screen to detect RubisCO enzymes even from organisms that would not be expected to be targeted.

Wheat wounding-responsive HD-Zip IV transcription factor GL7 is predominantly expressed in grain and activates genes encoding defensins.

Several classes of transcription factors are involved in the activation of defensins. A new type of the transcription factor responsible for the regulation of wheat grain specific defensins was characterised in this work. HD-Zip class IV transcription factors constitute a family of multidomain proteins. A full-length cDNA of HD-Zip IV, designated TaGL7 was isolated from the developing grain of bread wheat, using a specific DNA sequence as bait in the Y1H screen. 3D models of TaGL7 HD complexed with DNA cis-elements rationalised differences that underlined accommodations of binding and non-binding DNA, while the START-like domain model predicted binding of lipidic molecules inside a concave hydrophobic cavity. The 3'-untranslated region of TaGL7 was used as a probe to isolate the genomic clone of TdGL7 from a BAC library prepared from durum wheat. The spatial and temporal activity of the TdGL7 promoter was tested in transgenic wheat, barley and rice. TdGL7 was expressed mostly in ovary at fertilisation and its promoter was active in a liquid endosperm during cellularisation and later in the endosperm transfer cells, aleurone, and starchy endosperm. The pattern of TdGL7 expression resembled that of genes that encode grain-specific lipid transfer proteins, particularly defensins. In addition, GL7 expression was upregulated by mechanical wounding, similarly to defensin genes. Co-bombardment of cultured wheat cells with TdGL7 driven by constitutive promoter and seven grain or root specific defensin promoters fused to GUS gene, revealed activation of four promoters. The data confirmed the previously proposed role of HD-Zip IV transcription factors in the regulation of genes that encode lipid transfer proteins involved in lipid transport and defence. The TdGL7 promoter could be used to engineer cereal grains with enhanced resistance to insects and fungal infections.

Effects of somatic embryogenesis on gene expression of cloned coffee heterozygous hybrids

Our analyses suggest that no major genome organization occurred during SE process implying the nonoccurrence of somaclonal variation. However, the genetic background determines the quality of the in vitro response. Cloning of superior coffee plants by somatic embryogenesis can assist breeding programs on reducing the cost and time for launch of new cultivars. This study aimed to evaluate the efficiency of this methodology for cloning coffee trees with high heterozygosity, and to gather evidence that clonal progenies are faithful copies of mother plants. Selected plants IAC1 and IAC 2 from Coffea arabica breeding populations, resistant to leaf rust and leaf miner, respectively, were cloned via indirect somatic embryogenesis. Expression of selected genes involved in biological processes potentially affected by in vitro cultivation was evaluated by quantitative analysis. Genes encoding proteins associated with maintenance of DNA integrity and control of cell cycle presented predictable expression patterns along the clonal multiplication process. There were differences in the expression pattern of genes linked to in vitro cultivation-related stress, which were observed comparing either IAC 1 and IAC2 genotypes or clones and their corresponding mother plant. Those analyses suggest that the somatic embryogenesis does not lead to major genomic instability and clones are identical copies of mother plants, even with detected differences in the expression of genes that influence the response of in vitro cultivation.

Development of a rapid, simple and efficient one-pot cloning method for a reverse genetics system of broad subtypes of influenza A virus

The reverse genetics (RG) system of influenza A viruses is well established. However, the conventional sequence-dependent method for cloning influenza genome segments is time-consuming and requires multiple processes (eg. enzyme digestion and ligation) and exhibits low cloning efficiency compared to the sequence-independent cloning method. In this study, we improved influenza genome cloning into the pHW2000 vector for an RG system by incorporating a sequence-independent circular polymerase extension cloning (CPEC) approach which requires only 2 steps (reverse transcription and one-pot CPEC-PCR) and takes about 4 hours before the transformation. The specifically designed viral gene and vector primers used for CPEC-PCR have improved cloning efficiency ranging from 63.6 to 100% based on the results of gene-specific colony PCR which was additionally confirmed by enzyme digestion. We successfully cloned all genes from broad subtypes of influenza A viruses (H1-H12, N1-N9) and rescued by the RG system. Our results demonstrate that this method—one-Pot cloning for influenza A virus—was efficient in terms of required time and cloning rate. In conclusion, the novel cloning method for influenza A virus will contribute to a significant reduction in the time required for genetic studies of emerging influenza viruses.

Identification and functional study of an iif2 gene cluster for indole degradation in Burkholderia sp. IDO3

Burkholderia sp. IDO3 is an indole-degrading bacterium isolated from activated sludge. A previous genomic clone library assay identified an iif1 gene cluster for indole metabolism in strain IDO3. To further explore the underlying indole degradation mechanisms, the complete genome of strain IDO3 was sequenced (8,003,806 bp). The genome contained three circular chromosomes and one plasmid, and 7550 genes were predicted. Interestingly, in addition to iif1 on chromosome 3, bioinformatic analyses identified a second indole oxygenase gene cluster, iif2, on chromosome 1. Both iif clusters were up-regulated in response to indole. Heterologous expression of iifC1D1 and iifC2D2 in Escherichia coli BL21(DE3) demonstrated that these genes were capable of oxidizing indole to indigo. Gene knockout assays provided additional evidence that iifC2 played crucial roles in indole metabolism. In addition, we identified a novel gene (iifF) in the iif2 cluster. This gene was shown to encode an isatin hydrolase. IifF was expressed in E. coli, and a purified his-tagged enzyme preparation was obtained. IifF converted isatin to isatinate with Km of 4.4 ± 0.7 μM and kcat of 95.5 ± 4 s−1. This is the first study to show that indole can be degraded by two iif gene clusters, and that isatin hydrolase is involved in indole metabolism, improving our understanding of indole metabolic processes.

Engineering Synthetic Chromosomes by Sequential Loading of Multiple Genomic Payloads over 100 Kilobase Pairs in Size

Gene delivery vehicles currently in the clinic for treatment of monogenic disorders lack sufficient carrying capacity to efficiently address complex polygenic diseases. Thus, to engineer multifaceted genetic circuits for bioengineering human cells as a therapeutic option for polygenic diseases, we require new tools that are currently in their infancy. Mammalian artificial chromosomes, or synthetic chromosomes, represent a viable approach for delivery of large genetic payloads that are mitotically stable and remain independent of the host genome. Previously, we described a mammalian synthetic chromosome platform, termed the ACE system, that requires a single unidirectional integrase for the introduction of multiple genes onto the ACE platform chromosome. In this report, we provide a proof of concept that the ACE synthetic chromosome bioengineering platform is amenable to sequential delivery of off-the-shelf large genomic fragments. Specifically, large genomic clones spanning the human solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1 or GLUT1, 169 kbp), and human monocarboxylate transporter 1 (SLC16A1 or MCT1, 144 kbp) genetic loci were engineered onto the ACE platform and demonstrated to express and correctly splice both gene transcripts. Thus, the ACE system provides a facile and tractable engineering platform for the development of gene-based therapeutic agents targeting polygenic diseases.

Novel subgenotype D11 of hepatitis B virus in NaPo County, Guangxi, bordering Vietnam.

Hepatitis B virus has been classified into 10 genotypes and 48 subgenotypes worldwide. We found previously, through polymerase chain reaction (PCR) amplification of a sample collected in 2011, that an HBsAg carrier was infected with two genotypes (B and D) of HBV. We carried out cloning, sequencing and phylogenetic analysis of the complete genomes and, for confirmation, analysed a sample collected from the same individual in 2018. Fifteen complete sequences were obtained from each sample. The carrier was infected in 2011 by genotypes B and D and by various recombinants, but only genotype D was present in 2018. The major and minor parents of the recombinants are genotypes B and D, respectively, although the recombination breakpoints vary among them. All 23 genotype D isolates form a cluster, branching out from other subgenotype D sequences and supported by a 100 % bootstrap value. Based on complete genome sequences, almost all of the estimated intragroup nucleotide divergence values between our isolates and HBV subgenotypes D1-D10 exceed 4 %. Compared to the other subgenotypes (D1-D10), 35 unique amino acids were present in our isolates. Our data provide evidence for a novel subgenotype, provisionally designated HBV subgenotype D11.

A real-time loop mediated isothermal amplification assay for molecular detection of Burkholderia mallei, the aetiological agent of a zoonotic and re-emerging disease glanders

Burkholderia mallei, a potential biological warfare agent, is the causative agent of an infectious, fatal, and zoonotic disease, called glanders. Accurate and early diagnosis of glanders is important to control the disease lethality and infection spread. Molecular detection of B. mallei is considered strenuous because B. mallei is a subtractive genomic clone of B. pseudomallei. The present study was aimed at development of a real-time LAMP assay for detection of B. mallei. The LAMP assay was highly sensitive and could detect ≥250 fg of genomic DNA of B. mallei and ≥100 copies of recombinant plasmid containing target DNA sequence. In artificially spiked blood and water samples, it could detect ≥2.1 × 103 and ≥4.73 × 102 CFU/mL of B. mallei, respectively. The assay was highly specific for B. mallei as none of the other bacteria used in the study tested positive. The reported LAMP assay being simple and rapid can be a viable alternative to PCR-based glanders diagnostic assays in glanders endemic regions with resource-limited settings.

Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants.

Near full genome sequencing (NFGS) of HIV-1 is required to assess the genetic composition of HIV-1 strains comprehensively. Population-wide, it enables a determination of the heterogeneity of HIV-1 and the emergence of novel/recombinant strains, while for each individual it constitutes a diagnostic instrument to assist targeted therapeutic measures against viral components. There is still a lack of robust and adaptable techniques for efficient NFGS from miscellaneous HIV-1 subtypes. Using rational primer design, a broad primer set was developed for the amplification and sequencing of diverse HIV-1 group M variants from plasma. Using pure subtypes as well as diverse, unique recombinant forms (URF), variable amplicon approaches were developed for NFGS comprising all functional genes. Twenty-three different genomes composed of subtypes A (A1), B, F (F2), G, CRF01_AE, CRF02_AG, and CRF22_01A1 were successfully determined. The NFGS approach was robust irrespective of viral loads (≥306 copies/mL) and amplification method. Third-generation sequencing (TGS), single genome amplification (SGA), cloning, and bulk sequencing yielded similar outcomes concerning subtype composition and recombinant breakpoint patterns. The introduction of a simple and versatile near full genome amplification, sequencing, and cloning method enables broad application in phylogenetic studies of diverse HIV-1 subtypes and can contribute to personalized HIV therapy and diagnosis.

Engineering Responses to Amino Acid Substitutions in the VP0- and VP3-Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O.

The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH4Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.

Discovery, characterisation and genomic variation of six novel Gammapapillomavirus types from penile swabs in South Africa

Six novel human papillomaviruses from penile swabs were characterised. Multiple full genome clones for each novel type were generated, and complete genome sizes were: HPV211 (7253bp), HPV212 (7208bp), HPV213 (7096bp), HPV214 (7357), HPV215 (7186bp) and HPV216 (7233bp). Phylogenetically the novel papillomaviruses all clustered with Gammapapillomaviruses: HPV211 is most closely related to HPV168 (72% identity in the L1 nucleotide sequence) of the Gamma-8 species, HPV212 is most closely related to HPV144 (82.9%) of the Gamma-17 species, HPV213 is most closely related to HPV153 (71.8%) of the Gamma-13 species, HPV214 is most closely related to HPV103 (75.3%) of the Gamma-6 species, HPV215 and HPV216 are most closely related to HPV129 (76.8% and 79.2% respectively) of the Gamma-9 species. The novel HPV types demonstrated the classical genomic organisation of Gammapapillomavirusess, with seven open reading frames (ORFs) encoding five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) proteins. Typical of Gammapapillomavirusess the novel types all lacked the E5 ORF and HPV214 also lacked the E6 ORF. HPV212 had nine unique variants, HPV213 had five and HPV215 had four variants. Conserved domains observed among the novel types are the Zinc finger Binding Domain and PDZ domains. A retinoblastoma binding domain (pRB) binding domain in E7 protein was additionally identified in HPV214. This study expands the knowledge of the rapidly growing Gammapapillomavirus genus.