Origin and Biology of Simian Immunodeficiency Virus in Wild-Living Western Gorillas

Western lowland gorillas (Gorilla gorilla gorilla) are infected with a simian immunodeficiency virus (SIVgor) that is closely related to chimpanzee and human immunodeficiency viruses (SIVcpz and HIV-1, respectively) in west central Africa. Although existing data suggest a chimpanzee origin for SIVgor, a paucity of available sequences has precluded definitive conclusions. Here, we report the molecular characterization of one partial (BQ664) and three full-length (CP684, CP2135, and CP2139) SIVgor genomes amplified from fecal RNAs of wild-living gorillas at two field sites in Cameroon. Phylogenetic analyses showed that all SIVgor strains clustered together, forming a monophyletic lineage throughout their genomes. Interestingly, the closest relatives of SIVgor were not SIVcpzPtt strains from west central African chimpanzees (Pan troglodytes troglodytes) but human viruses belonging to HIV-1 group O. In trees derived from most genomic regions, SIVgor and HIV-1 group O formed a sister clade to the SIVcpzPtt lineage. However, in a tree derived from 5' pol sequences (similar to 900 bp), SIVgor and HIV-1 group O fell within the SIVcpzPtt radiation. The latter was due to two SIVcpzPtt strains that contained mosaic pol sequences, pointing to the existence of a divergent SIVcpzPtt lineage that gave rise to SIVgor and HIV-1 group O. Gorillas appear to have acquired this lineage at least 100 to 200 years ago. To examine the biological properties of SIVgor, we synthesized a full-length provirus from fecal consensus sequences. Transfection of the resulting clone (CP2139.287) into 293T cells yielded infectious virus that replicated efficiently in both human and chimpanzee CD4(+) T cells and used CCR5 as the coreceptor for viral entry. Together, these results provide strong evidence that P. t. troglodytes apes were the source of SIVgor. These same apes may also have spawned the group O epidemic; however, the possibility that gorillas served as an intermediary host cannot be excluded

Chimpanzee reservoirs of pandemic and nonpandemic HIV-1

Human immunodeficiency virus type 1 (HIV-1), the cause of human acquired immunodeficiency syndrome ( AIDS), is a zoonotic infection of staggering proportions and social impact. Yet uncertainty persists regarding its natural reservoir. The virus most closely related to HIV-1 is a simian immunodeficiency virus ( SIV) thus far identified only in captive members of the chimpanzee subspecies Pan troglodytes troglodytes. Here we report the detection of SIVcpz antibodies and nucleic acids in fecal samples from wild-living P.t. troglodytes apes in southern Cameroon, where prevalence rates in some communities reached 29 to 35%. By sequence analysis of endemic SIVcpz strains, we could trace the origins of pandemic ( group M) and nonpandemic ( group N) HIV-1 to distinct, geographically isolated chimpanzee communities. These findings establish P. t. troglodytes as a natural reservoir of HIV-1

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-017-0206-0) contains supplementary material, which is available to authorized users

Influence of primer & probe chemistry and amplification target on reverse transcription digital PCR quantification of viral RNA

Compared to other PCR technologies, digital PCR is a potentially highly accurate approach for the quantification of nucleic acid fragments. This study describes the impact of four experimental factors, namely primer and probe chemistry, PCR amplification target, duplexing, and template type, on the measurement results obtained by reverse transcription digital PCR (RT-dPCR) of viral RNA using influenza A virus as a model. Along conventional dual labelled probes (DLP), alternative primer and probe chemistries, including Zip Nucleic Acids (ZNAs), Locked Nucleic Acids (LNAs), and Scorpions®, were compared with two RNA template types: i) total genomic RNA extracted from cell cultured influenza A and ii) a synthetically prepared RNA transcript (In vitro transcribed RNA). While apparently duplexing or a different PCR target choice did not have a significant influence on the estimated RNA copy numbers, the impact of the choice of primer and probe chemistry and template type differed significantly for some methods. The combined standard uncertainty of the dPCR analysis results has been assessed, taking into account both the repeatability and the intermediate precision of the procedure. Our data highlight the importance of dPCR method optimisation and the advantage of using a more sophisticated primer and probe chemistry, which turned out to be dependent on the template type. Considerations are provided with respect to the molecular diagnostics of viral RNA pathogens, and more specifically, for precise quantification of RNA, which is of tremendous importance for the development of RNA calibration materials and the qualification of these calibrants as certified reference materials

CERTIFICATION REPORT: The Certification of the PFGE fragment sizes of Listeria monocytogenes (strain H2446) DNA in agarose plugs: ERM®-AD624

Summary This report describes the production of ERM®-AD624, a Listeria monocytogenes DNA material certified for the size of the DNA fragments obtained by enzymatic restriction digestion and Pulsed Field Gel Electrophoresis (PFGE). This material was produced following ISO Guide 34:2009 [ ] and is certified in accordance with ISO Guide 35:2006 [ ]. The CRM was produced from a culture of Listeria monocytogenes strain H2446 and processed into agarose plugs suitable for PFGE. The bacteria were lysed to release the DNA within the plugs. Between unit-homogeneity and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The material was characterised by an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025. Technically invalid results were removed but no outliers were eliminated on statistical grounds only. Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM). The material is intended for quality control and assessment of method performance. As with any reference material, it can be used for establishing control charts or validation studies. The CRM is available in plastic screw cap vials containing one plug suspended in TE solution. The minimum amount of sample to be used is the whole CRM.JRC.F.6-Reference Material

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA

Quantitative PCR (qPCR) is an important tool in pathogen detection; however, the use of different qPCR components, calibration materials and DNA extraction methods reduces the comparability between clinics, which could result in false diagnosis and discrepancies in patient care. The establishment of a metrological framework for nucleic-acid tests is expected to improve the degree of standardisation of pathogen detection and quantification methods applied in a clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) allows quantification of nucleic acids and has already been used for a myriad of applications, including pathogen quantification. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessments on its actual performance characteristics should be collected before it can be implemented in a metrological framework and to allow an adequate estimation of the measurement uncertainty. Here, high repeatability and reproducibility of dPCR for quantification of DNA from human cytomegalovirus were demonstrated. Using extracted DNA and whole-virus material, each of five dPCR platforms from four laboratories demonstrated high intermediate precision between three consecutive experiments. Furthermore, discrepancies in estimated mean DNA copy-number concentrations between different laboratories were less than two-fold, with DNA extraction recognised as the main source of variability. Our results demonstrate dPCR-based methods can be very repeatable and reproducible for quantification of viral DNA, and should be considered as potent reference method candidates for implementation in a metrological framework.JRC.F.6-Reference Material