Validation of specific quantitative real-time RT-PCR assay panel for Infectious Bronchitis using synthetic DNA standards and clinical specimens.

Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan™-based quantitative real-time RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan™-based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log10 dynamic range with a reproducible limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%-115%. Further validation of specificity using clinical and biological specimens was also successful.

DNA assembly with error correction on a droplet digital microfluidics platform.

BACKGROUND: Custom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform. RESULTS: We optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 µL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl2, Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb- 1. CONCLUSION: We demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization.

Xenogenic rolling-circle replication of a synthetic beak and feather disease virus genomic clone in 293TT mammalian cells and Nicotiana benthamiana.

The preparation of infectious beak and feather disease circovirus virions (BFDV) has until now relied on the extraction of virus from whole tissue of deceased or euthanized parrots known to be infected with the virus. Extraction from diseased tissue is necessary, as the virus has yet to be grown in vitro using tissue-cultured cells from any source. While infectious DNA clones have been synthesized for porcine and duck circoviruses, and both replicate in host cells and result in active viral infection in animals, this has not been shown for BFDV. The aim of this study was to prepare an infectious BFDV genomic clone that could be used as challenge material in birds for vaccine testing. A putatively infectious BFDV genomic clone was designed and tested in mammalian cell culture, and in the plant Nicotiana benthamiana in the presence of plant-specific ssDNA geminivirus replication components. Replication was assessed using rolling-circle amplification, qPCR, replication-deficient clones and rescue plasmids. We showed that a synthetic partially dimeric BFDV genomic clone self-replicated when transfected into 293TT mammalian cells, and was also replicated in N. benthamiana in the presence of geminivirus replication elements. This is the first report of a BFDV genome replicating in any cell system, and the first report of a circovirus replicating with the aid of a geminivirus in a plant. Both of these developments could open up possibilities for making reagents and vaccines for BFDV, testing vaccine efficacy and investigating viral replication using rationally designed artificial genomes.

In vitro and in vivo replication of a chemically synthesized consensus genome of hepatitis B virus genotype B.

Hepatitis B virus (HBV) genotypes vary in their geographical distribution and virological features. Previous investigations have shown that HBV genotype B is a predominant HBV genotype in China. Studies on HBV concerning different isolates frequently meet the question about the HBV reference strain and its representativeness. Although HBV consensus sequences can be generated easily by sequence alignment, they may not exist in nature or could not usually be isolated from patient samples. Thus, the construction of a consensus HBV genome has been proposed. In this study, an HBV genotype B consensus sequence was established by comparing 42 full-length HBV genotype B sequences and the genome was generated by chemical synthesis. This consensus genome was fully replication competent by in vitro transfection into hepatoma cells. The plasmid pHBV1.3B carrying a 1.3× full-length HBV consensus genome was hydrodynamically injected into Balb/c mice. HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc detection indicated expression and replication of this HBV genome in mice, similar to other HBV isolates. This approach represents a strategy to design and create consensus HBV genomes for future studies.

Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

BACKGROUND: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts. METHODS: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species. RESULTS: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe. CONCLUSIONS: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

Toward larger DNA origami.

Structural DNA nanotechnology, and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7,249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Additionally, increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) production of the largest to-date biologically derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have experimentally demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addition, we have demonstrated two-dimensional, asymmetric origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by atomic force microscopy.

Proof by synthesis of Tobacco mosaic virus.

BACKGROUND: Synthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras. RESULTS: RNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana. CONCLUSIONS: This first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly.

A simple, universal, efficient PCR-based gene synthesis method: sequential OE-PCR gene synthesis.

Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.

A synthetic codon-optimized hepatitis C virus nonstructural 5A DNA vaccine primes polyfunctional CD8+ T cell responses in wild-type and NS5A-transgenic mice.

The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >10(4) postimmunization. With respect to CD8(+) T cell responses, the coNS5A gene primed more potent IFN-γ-producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8(+) T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8(+) T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.

Synthetic poliovirus and other designer viruses: what have we learned from them?

Owing to known genome sequences, modern strategies of DNA synthesis have made it possible to recreate in principle all known viruses independent of natural templates. We describe the first synthesis of a virus (poliovirus) in 2002 that was accomplished outside living cells. We comment on the reaction of laypeople and scientists to the work, which shaped the response to de novo syntheses of other viruses. We discuss those viruses that have been synthesized since 2002, among them viruses whose precise genome sequence had to be established by painstakingly stitching together pieces of sequence information, and viruses involved in zoonosis. Synthesizing viral genomes provides a powerful tool for studying gene function and the pathogenic potential of these organisms. It also allows modification of viral genomes to an extent hitherto unthinkable. Recoding of poliovirus and influenza virus to develop new vaccine candidates and refactoring the phage T7 DNA genome are discussed as examples.

Total chemical synthesis, assembly of human torque teno virus genome.

Torque teno virus (TTV) is a nonenveloped virus containing a single-stranded, circular DNA genome of approximately 3.8kb. We completely synthesized the 3 808 nucleotides of the TTV (SANBAN isolate) genome, which contains a hairpin structure and a GC-rich region. More than 100 overlapping oligonucleotides were chemically synthesized and assembled by polymerase chain assembly reaction (PCA), and the synthesis was completed with splicing by overlap extension (SOEing). This study establishes the methodological basis of the chemical synthesis of a viral genome for use as a live attenuated vaccine or gene therapy vector.

Evaluation of norovirus removal performance in a coagulation-ceramic microfiltration process by using recombinant norovirus virus-like particles.

Norovirus (NV) is a prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis worldwide. Because of the lack of a cell culture system or an animal model for this virus, studies on drinking water treatment such as separation and disinfection processes are still hampered. In the present study, we investigated NV removal performance as particles during a coagulation-ceramic microfiltration (MF) process by using recombinant NV virus-like particles (rNV-VLPs), which are morphologically and antigenically similar to native NV. We also experimentally investigated the behaviors of two widely accepted surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, for comparison with the behavior of rNV-VLPs. More than 4-log removal was observed for rNV-VLPs with a 1.08 mg-Al/L dose of polyaluminium chloride in the coagulation-ceramic MF process. This high removal ratio of rNV-VLPs satisfies the U.S. Environmental Protection Agency requirement of 4-log removal or inactivation. In addition, the removal ratios of Qbeta and MS2 were approximately 2-log and 1-log, smaller than the ratio of rNV-VLPs. Accordingly, both bacteriophages have the potential to become appropriate surrogates for native NV in the coagulation-ceramic MF process, and, of the two, Qbeta is the more conservative surrogate.

A randomized lentivirus shRNA library construction.

Vector based shRNA (short hairpin RNA) expression library has been widely used to screen functional genes. For two main methods that have been used to generate short hairpin RNA libraries, chemical synthesis is too expensive to be widely used and the low efficiency of enzymatic approach makes it difficult to construct. We have developed a protocol to construct a new kind of shRNA library called randomized shRNA library. Within three steps chemically synthesized randomized 19-mers DNA were efficiently converted to double-stranded DNA fragments containing shRNA templates. This kind of shRNA library permits simple and economic construction, providing another choice for whole-genome phenotypic screening of genes.

The effect of backbone structure on polycation comb-type copolymer/DNA interactions and the molecular assembly of DNA.

A series of comb-type copolymers comprised of various polycation backbones and dextran (Dex) side chains were prepared to study the DNA/copolymer interaction. While the cationic copolymers with a lower degree of dextran grafts maintained an ability to condense DNA molecules into a globule form those with a higher degree of dextran grafting interacted with DNA without inducing DNA condensation. The structural differences in cationic backbones diversely influenced DNA hybridization as evaluated by circular dichroism (CD) spectrometry and UV-melting analyses. The copolymer having a polyallylamine (PAA) backbone induced B-->A-type transformation of DNA duplex, whereas the copolymers having either alpha-poly(l-lysine) (alpha PLL) or epsilon-poly(l-lysine) (epsilon PLL) backbone induced B-->C-type transformation. The PAA copolymer is the first example of the artificial polymer that induces B-->A-type transformation under physiologically relevant condition. UV-melting analyses of DNA strands indicated that the alpha PLL copolymers showed the highest stabilization efficacy toward poly(dA).poly(dT) duplex and poly(dA).2poly(dT) triplex without affecting reversibility of inter DNA association. Melting temperatures (T(m)) of the triplex increased from 38 degrees Celsius to 99 degrees Celsius by the addition of the alpha PLL copolymer with an appropriate grafting degree. While the PAA copolymers had higher density of cationic groups along the backbone than alpha PLL copolymers, these copolymers moderately increased T(m) of the DNA triplex. The PAA copolymer caused considerable hysteresis in thermal melting/reassociation processes. Note that the PLL copolymers increased T(m) of the DNA triplex and not the duplex, suggesting their potential as a triplex selective stabilizer. Chemical structures of the cationic backbones of the copolymers were characteristically affected on the copolymer/DNA interaction even if their backbones were surrounded by abundant side chains (> wt%) of dextran. The study suggested that tailor-made design of "functional polycounterion" is a strategy to engineer molecular assembling of DNA.

Lysine directed cross-linking of viral DNA-RNA:DNA hybrid substrate to the isolated RNase H domain of HIV-1 reverse transcriptase.

An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA-RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn(2+). The Mn(2+) titration of cross-linking paralleled the Mn(2+) requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H-nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.

Exponential DNA replication by laminar convection.

It is shown that laminar thermal convection can drive a chain reaction of DNA replication. The convection is triggered by a constant horizontal temperature gradient, moving molecules along stationary paths between hot and cold regions. This implements the temperature cycling for the classical polymerase chain reaction (PCR). The amplification is shown to be exponential and reaches 100,000-fold gains within 25 min. Besides direct applications, the mechanism might have implications for the molecular evolution of life.