Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens

We developed a conceptually new subtraction strategy for the detection and isolation of target DNA and/or RNA from complex nucleic acid mixtures, called Primer Extension Enrichment Reaction (PEER). PEER uses adapters and class IIS restriction enzymes to generate tagged oligonucleotides from dsDNA fragments derived from specimens containing an unknown target (‘tester’). Subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddNTPs and a double stranded DNA template generated from a pool of normal specimens (‘driver’). Primers that do not acquire ddNTP are used to capture and amplify the unique target DNA from the original tester dsDNA. We successfully applied PEER to specimens containing known infectious agents (Hepatitis B Virus and Walrus Calicivirus) and demonstrated that it has higher efficiency than the best comparable technique. The strategy used for PEER is versatile and can be adapted for the identification of known and unknown pathogens and mutations, differential expression studies and other applications that allow the use of subtractive strategies

Experimentelle Kaninchensyphilis und das Reticuloendothelialsystem

Bei experimenteller Kaninchensyphilis fuhrte der Verfasser seine Versuche aus, berucksichtigt auf die klinischen Befunde und die Wassermannsche Reaktion des mit Sp. pallida geimpften Kaninchens bei der Splenektomie, bei der Blockierung des Reticuloendothelialsystemes und bei der Einspritzung des Milzextractes. Die Resultate sind die folgenden: 1. Nachdem Spirochaeta pallida dem Kaninchen in den Hoden geimpft wurde, wurde die Blockierung mit Tusche ausgefuhrt. Im Verlaufe der Infektion zeigten sich lokale Befunde und Wassermannsche Reaktion die in jeder Hinsicht denen der Kontrollen glichen. 2. Beim Kaninchen, welches gleichzeizig entmilzt und mit Sp. pallida geimpft wurde, trat der Lokalaffekt etwas fruhzeizig auf und die Wassermannsche Reaktion war kurzdauernd positiv. 3. Falls die Impfung nach der Entmilzung ausgefuhrt war, stimmten die Resultate fast mit den obigen ein. 4. Wenn die Versuchstiere erst geimpft und dann entmilzt wurden, so wurde eine bemerkenswerte oedematose Anschwellung des Hodensacks und ein Kurzdauernder positive Ausfall der Wa. R. beobachtet. 5. Wenn die Entmilzung, Impfung und Blockierung gleichzeizig ausgefuhrt wurden, so zeigten sich die lokalen sowie metastatischen Befunde bei Versuchstieren wie ohne Entmilzung, doch war die positive Phase der Wa. R. bedeutend verkurzt und dabei ihre Grad niederwertig. 6. Erst geimpft und dann mit Milzextract wiederhohlt gespritzt, so verliefen die lokalen sowie metastatischen Befunde leichter und die Wa. R. brach fruhzeizig positiv aus und dauerte langer. (Autoreferat.

Evaluation of Intra-Host Variants of the Entire Hepatitis B Virus Genome

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings

Epidemic History and Evolutionary Dynamics of Hepatitis B Virus Infection in Two Remote Communities in Rural Nigeria

BACKGROUND: In Nigeria, hepatitis B virus (HBV) infection has reached hyperendemic levels and its nature and origin have been described as a puzzle. In this study, we investigated the molecular epidemiology and epidemic history of HBV infection in two semi-isolated rural communities in North/Central Nigeria. It was expected that only a few, if any, HBV strains could have been introduced and effectively transmitted among these residents, reflecting limited contacts of these communities with the general population in the country. METHODS AND FINDINGS: Despite remoteness and isolation, approximately 11% of the entire population in these communities was HBV-DNA seropositive. Analyses of the S-gene sequences obtained from 55 HBV-seropositive individuals showed the circulation of 37 distinct HBV variants. These HBV isolates belong predominantly to genotype E (HBV/E) (n=53, 96.4%), with only 2 classified as sub-genotype A3 (HBV/A3). Phylogenetic analysis showed extensive intermixing between HBV/E variants identified in these communities and different countries in Africa. Quasispecies analysis of 22 HBV/E strains using end-point limiting-dilution real-time PCR, sequencing and median joining networks showed extensive intra-host heterogeneity and inter-host variant sharing. To investigate events that resulted in such remarkable HBV/E diversity, HBV full-size genome sequences were obtained from 47 HBV/E infected persons and P gene was subjected to Bayesian coalescent analysis. The time to the most recent common ancestor (tMRCA) for these HBV/E variants was estimated to be year 1952 (95% highest posterior density (95% HPD): 1927-1970). Using additional HBV/E sequences from other African countries, the tMRCA was estimated to be year 1948 (95% HPD: 1924-1966), indicating that HBV/E in these remote communities has a similar time of origin with multiple HBV/E variants broadly circulating in West/Central Africa. Phylogenetic analysis and statistical neutrality tests suggested rapid HBV/E population expansion. Additionally, skyline plot analysis showed an increase in the size of the HBV/E-infected population over the last approximately 30-40 years. CONCLUSIONS: Our data suggest a massive introduction and relatively recent HBV/E expansion in the human population in Africa. Collectively, these data show a significant shift in the HBV/E epidemic dynamics in Africa over the last century

Characteristics of Adults in the Hepatitis B Research Network in North America Reflect Their Country of Origin and Hepatitis B Virus Genotype

Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma worldwide; populations that migrate to the US and Canada might be disproportionately affected. The Hepatitis B Research Network (HBRN) is a cooperative network of investigators from the United States and Canada, created to facilitate clinical, therapeutic, and translational research in adults and children with hepatitis B. We describe the structure of the network and baseline characteristics of adults with hepatitis B enrolled in the network

Primer Extension Enrichment Reaction (PEER):

a new subtraction method for identification of genetic differences between biological specimen

Hepatitis B Virus and Hepatitis C Virus Infections in United States-Bound Refugees from Asia and Africa

Photograph of a scene with Great Plains Chautauqua reenactors

Automated quality control for a molecular surveillance system

Abstract Background Molecular surveillance and outbreak investigation are important for elimination of hepatitis C virus (HCV) infection in the United States. A web-based system, Global Hepatitis Outbreak and Surveillance Technology (GHOST), has been developed using Illumina MiSeq-based amplicon sequence data derived from the HCV E1/E2-junction genomic region to enable public health institutions to conduct cost-effective and accurate molecular surveillance, outbreak detection and strain characterization. However, as there are many factors that could impact input data quality to which the GHOST system is not completely immune, accuracy of epidemiological inferences generated by GHOST may be affected. Here, we analyze the data submitted to the GHOST system during its pilot phase to assess the nature of the data and to identify common quality concerns that can be detected and corrected automatically. Results The GHOST quality control filters were individually examined, and quality failure rates were measured for all samples, including negative controls. New filters were developed and introduced to detect primer dimers, loss of specimen-specific product, or short products. The genotyping tool was adjusted to improve the accuracy of subtype calls. The identification of “chordless” cycles in a transmission network from data generated with known laboratory-based quality concerns allowed for further improvement of transmission detection by GHOST in surveillance settings. Parameters derived to detect actionable common quality control anomalies were incorporated into the automatic quality control module that rejects data depending on the magnitude of a quality problem, and warns and guides users in performing correctional actions. The guiding responses generated by the system are tailored to the GHOST laboratory protocol. Conclusions Several new quality control problems were identified in MiSeq data submitted to GHOST and used to improve protection of the system from erroneous data and users from erroneous inferences. The GHOST system was upgraded to include identification of causes of erroneous data and recommendation of corrective actions to laboratory users

HCV transmission in high-risk communities in Bulgaria.

BACKGROUND:The rate of HIV infection in Bulgaria is low. However, the rate of HCV-HIV-coinfection and HCV infection is high, especially among high-risk communities. The molecular epidemiology of those infections has not been studied before. METHODS:Consensus Sanger sequences of HVR1 and NS5B from 125 cases of HIV/HCV coinfections, collected during 2010-2014 in 15 different Bulgarian cities, were used for preliminary phylogenetic evaluation. Next-generation sequencing (NGS) data of the hypervariable region 1 (HVR1) analyzed via the Global Hepatitis Outbreak and Surveillance Technology (GHOST) were used to evaluate genetic heterogeneity and possible transmission linkages. Links between pairs that were below and above the established genetic distance threshold, indicative of transmission, were further examined by generating k-step networks. RESULTS:Preliminary genetic analyses showed predominance of HCV genotype 1a (54%), followed by 1b (20.8%), 2a (1.4%), 3a (22.3%) and 4a (1.4%), indicating ongoing transmission of many HCV strains of different genotypes. NGS of HVR1 from 72 cases showed significant genetic heterogeneity of intra-host HCV populations, with 5 cases being infected with 2 different genotypes or subtypes and 6 cases being infected with 2 strains of same subtype. GHOST revealed 8 transmission clusters involving 30 cases (41.7%), indicating a high rate of transmission. Four transmission clusters were found in Sofia, three in Plovdiv, and one in Peshtera. The main risk factor for the clusters was injection drug use. Close genetic proximity among HCV strains from the 3 Sofia clusters, and between HCV strains from Peshtera and one of the two Plovdiv clusters confirms a long and extensive transmission history of these strains in Bulgaria. CONCLUSIONS:Identification of several HCV genotypes and many HCV strains suggests a frequent introduction of HCV to the studied high-risk communities. GHOST detected a broad transmission network, which sustains circulation of several HCV strains since their early introduction in the 3 cities. This is the first report on the molecular epidemiology of HIV/HCV coinfections in Bulgaria