Detection and differentiation of murine leukemia virus (MLV) and murine stem cell virus (MSCV) and therefrom derived nucleic acids.

Murine leukemia virus (MLV) and murine stem cell virus (MSCV) and derived retroviral vectors are widely used to study retrovirus biology and as tools for gene delivery. The method described here represents a quantitative real time PCR (qPCR) with hydrolysis probe that can be applied within classical qPCR as well as in digital droplet PCR (ddPCR). The method targets a 60 bp long fragment located within the U5 region of the MLV/MSCV genome sequence. For the here described method a LOD95% of 25 copies per PCR reaction (DNA) and 80 copies per PCR reaction (RNA) was determined, and PCR efficiencies of 92.5 % and 98.5 %, respectively, were observed. This method enables the fast and simple titration of viral genomic RNA present in retroviral vector stocks for accurate and consistent transduction experiments. Furthermore, it enables the detection of proviral and transfer plasmid derived DNA sequences and can be modified to differentiate between retroviral RNA and DNA.

Investigation of three outbreaks of Porcine Epidemic Diarrhea in Germany in 2016 demonstrates age dependent differences in the development of humoral immune response.

Porcine epidemic diarrhea (PED) has reemerged in Europe since 2014. Characterized by a rapid onset of diarrhea in pigs of all ages, morbidity can reach up to 100% whereas mortality is variable. The virus strains involved in the recent European outbreaks all cluster together with US strains (S INDEL) that lead to less severe clinical signs. In this study, fattening pigs and suckling piglets (n = 105) on farms with no prior PED history were monitored after an acute outbreak of the disease, caused by an S INDEL strain of PED virus (PEDV). For diagnostic investigations in the affected farms, real time RT-PCR was performed to detect PEDV RNA in individually taken fecal samples, and two commercial ELISA kits, both based on the N protein of PEDV, were used to detect IgG in serum samples of pigs experiencing acute signs of the disease. PEDV RNA could be detected in fecal samples up to 14 days after initial sampling. Comparing both ELISAs by Cohens Kappa showed substantial agreement (κ = 0,771). Antibodies were detectable in all fattening pigs (100%) within 10 days after the occurrence of first clinical signs and remained detectable for about two months at least in 20.6% (farm 1) and 45.7% (farm 2) of the animals, respectively. In contrast, only 18 of 34 (52.9%) suckling piglets seroconverted. Although, PEDV RNA was found in fecal samples of all piglets, 13 piglets did not demonstrate antibodies at any sampling day. PCR to detect PEDV RNA in fecal samples seems to be a reliable diagnostic tool during and after the acute outbreak. In the present study, IgG ELISA kits proved to be a feasible diagnostic tool, but age dependent differences in detection rate and persistence of antibodies need to be considered.

Reverse Transcription Quantitative Polymerase Chain Reaction for Detection of and Differentiation Between RNA and DNA of HIV-1-Based Lentiviral Vectors.

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

Strategies and methods for the detection and identification of viral vectors.

The production and application of viral vectors are frequently performed genetic engineering operations. HIV-1-based lentiviral vectors, AAV2-based, and adenoviral vectors are amongst the most abundant viral vectors utilized for gene delivery. They are generally classified into risk group 1 or 2 (according to EU directive 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work).

The Hepatitis E virus intraviral interactome.

Hepatitis E virus (HEV) is an emerging virus causing epidemic acute hepatitis in developing countries as well as sporadic cases in industrialized countries. The life cycle of HEV is still poorly understood and the lack of efficient cell culture systems and animal models are the principal limitations for a detailed study of the viral replication cycle. Here we exhaustively examine all possible intraviral protein-protein interactions (PPIs) of HEV by systematic Yeast two-hybrid (Y2H) and LuMPIS screens, providing a basis for studying the function of these proteins in the viral replication cycle. Key PPIs correlate with the already published HEV 3D structure. Furthermore, we report 20 novel PPIs including the homodimerization of the RNA dependent RNA polymerase (RdRp), the self-interaction of the papain like protease, and ORF3 interactions with the papain-like protease and putative replicase components: RdRp, methylase and helicase. Furthermore, we determined the dissociation constant (Kd) of ORF3 interactions with the viral helicase, papain-like protease and methylase, which suggest a regulatory function for ORF3 in orchestrating the formation of the replicase complex. These interactions may represent new targets for antiviral drugs.

Standard free droplet digital polymerase chain reaction as a new tool for the quality control of high-capacity adenoviral vectors in small-scale preparations.

High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy.

RsfA (YbeB) proteins are conserved ribosomal silencing factors.

The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells.

Matrix-based yeast two-hybrid screen strategies and comparison of systems.

Today, matrix-based screens are used primarily for smaller and medium-size clone collections in combination with automation and cloning techniques that allow for reliable and fast interaction screening. Matrix-based yeast two-hybrid screens are an alternative to library-based screens. However, intermediary forms are possible too and we compare both strategies, including a detailed discussion of matrix-based screens. Recent improvement of matrix screens (also called array screens) uses various pooling strategies as well as novel vectors that increase their efficiency while decreasing false-negative rates and increasing reliability.

Array-based yeast two-hybrid screens: a practical guide.

Yeast two-hybrid screens are carried out as random library screens or matrix-based screens. The latter have the advantage of being better controlled and thus typically give clearer results. In this chapter, we provide detailed protocols for matrix-based Y2H screens and give some helpful instructions how to plan a large-scale interaction screen. We also discuss strategies to identify or avoid false negatives and false positives.

Yeast two-hybrid screens: improvement of array-based screening results by N- and C-terminally tagged fusion proteins.

Matrix-based yeast two-hybrid screens are an alternative to library-based screens. Recent improvements of matrix screens (also called array screens), use various pooling strategies as well as novel vectors that increase its efficiency while decreasing the false-negative rate, thus increasing reliability. In this chapter, we describe a screening strategy that systematically combines N- and C-terminal fusion proteins using a recently developed vector system.

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors.

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome.

BACKGROUND: Yeast two-hybrid (Y2H) screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem. RESULTS: We have constructed two new vectors (pGBKCg and pGADCg) that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all approximately 4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV) proteins for interactions, using all possible combinations. About approximately 20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN) to 43% (CN-CC). Performing four screens (i.e. permutations) instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors. CONCLUSIONS: Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H interactions that can provide a measure of reproducibility without additional assays.

Evolutionarily conserved herpesviral protein interaction networks.

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.