Bovine viral diarrhea virus genomic variation within persistently infected cattle.

Bovine viral diarrhea virus (BVDV) is a single stranded RNA virus in the family Flaviviridae that causes a form of persistent infection. If a fetus is infected in utero during the first 120days of gestation the resulting calf will be immunotolerant to the infecting strain and maintain the virus for life. These animals are epidemiologically important in maintaining BVDV on farms, but also present a unique opportunity to study quasispecies in vivo in the absence of significant selection by the host adaptive immune response. We used deep sequencing and novel analytical methods to characterize the viral populations within the mesenteric lymph nodes of 10 persistently infected animals. Our results indicate that the pattern of variability across the viral genome from animal to animal is very consistent within BVDV subgenotypes. However, the individual mutations that constitute this variation are not necessarily the same in each animal. Even in the absence of significant immune selection the structural genes of BVDV vary more extensively than the non-structural genes. These findings could be useful for future vaccine design against BVDV as well as for measuring and understanding patterns of variation in other ssRNA viruses, especially those that belong to the family Flaviviridae.

Deep sequencing shows low-level oncogenic hepatitis B virus variants persists post-liver transplant despite potent anti-HBV prophylaxis.

Recent studies suggest that withdrawal of hepatitis B immune globulin (HBIG) and nucleos(t)ide analogues (NA) prophylaxis may be considered in HBV surface antigen (HBsAg)-negative liver transplant (LT) recipients with a low risk of disease recurrence. However, the frequency of occult HBV infection (OBI) and HBV variants after LT in the current era of potent NA therapy is unknown. Twelve LT recipients on prophylaxis were tested in matched plasma and peripheral blood mononuclear cells (PBMCs) for HBV quasispecies by in-house nested PCR and next-generation sequencing of amplicons. HBV covalently closed circular DNA (cccDNA) was detected in Hirt DNA isolated from PBMCs with cccDNA-specific primers and confirmed by nucleic acid hybridization and Sanger sequencing. HBV mRNA in PBMC was detected with reverse-transcriptase nested PCR. In LT recipients on immunosuppressive therapy (10/12 male; median age 57.5 [IQR: 39.8-66.5]; median follow-up post-LT 60 months; 6 pre-LT hepatocellular carcinoma [HCC]), 9 were HBsAg-. HBV DNA was detected in all plasma and PBMC tested; cccDNA and/or mRNA was detected in the PBMC of 10/12 patients. Significant HBV quasispecies diversity (ie 143-2212 nonredundant HBV species) was noted in both sites, and single nucleotide polymorphisms associated with cirrhosis and HCC were detected at varying frequencies. In conclusion, OBI and HBV variants associated with severe liver disease persist in LT recipients on prophylaxis. Although HBV control and cccDNA transcriptional silencing may occur despite immunosuppression, complete virological eradication does not occur in LT recipients with a history of HBV-related end-stage liver disease.

Optimizing gDNA extraction from fresh frozen meningioma tissue for downstream genetic analysis.

OBJECTIVE: Meningioma is the most common brain tumor. Genetic mutations in meningioma that include deletion of the neurofibromatosis type 2 gene, (NF2), offer diagnostic information on tumor behavior, recurrence and potential response to treatment. Obtaining high-grade genetic material is critical for accurate, sensitive and robust molecular testing. Currently, no standardized procedure exists for extracting gDNA from meningioma, and this problem was addressed in this report. METHOD: This study compared the yield and quality of extracted gDNA from patient meningioma specimens using an optimized phenol chloroform method and two commercial silica column-based extractions kits and tested respective performances as template in qPCR tests and multiplex ligation-dependent probe amplification (MLPA) NF2 screening. RESULTS: Mean gDNA yields were comparable for each method tested; however, phenol chloroform extraction outperformed column-based kits in all other quality assurance metrics examined. Phenol chloroform extracted gDNA was highly pure, and of a higher fragment size species when compared to column prepared gDNA. qPCR of GAPDH, B2MG, and RPL37A housekeeping genes demonstrated variance in cycle thresholds between patient samples was much lower in the phenol chloroform group. Similarly, primer efficiencies were significantly improved in this sample group which translated to a broader qPCR linear dynamic range and much improved qPCR performance at low concentrations of template. MLPA screening identified NF2 gene deletions in 6 of 12 meningioma samples. Inconsistencies in copy number data for NF2 and reference regions of the genome were observed between gDNA sample extraction groups that included both false negative and positive errors in silica column derived gDNA samples. CONCLUSIONS: This study outlines a highly robust phenol chloroform extraction method for obtaining high-quality gDNA from frozen meningioma tissue and highlights the significance of performing adequate quality assurance when using gDNA for downstream genetic analysis. Most importantly, we demonstrate using gDNA extracted with silica column based kits can lead to diagnostic errors when screening NF2 deletions in meningiomas with MLPA.

Loop mediated isothermal amplification (LAMP) for the detection and subtyping of human papillomaviruses (HPV) in oropharyngeal squamous cell carcinoma (OPSCC).

BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is a growing problem that presents a significant challenge to Otolaryngologist-Head and Neck Surgeons. Knowledge of HPV status yields critical prognostic information, with potential for treatment selection based on tumour HPV status. The current gold standard of diagnosis, PCR, is expensive, demanding and time consuming. Alternatives such as p16 immunohistochemistry are subjective and potentially inaccurate. Loop-mediated isothermal amplification (LAMP) is a rapid, robust and inexpensive molecular diagnostic technique. OBJECTIVES: Our aim was to verify LAMP as a potential bedside diagnostic assay for subtyping of HPV in OPSCC. STUDY DESIGN: DNA from 72 formalin-fixed paraffin embedded (FFPE) OPSCC patient samples was tested. PCR and LAMP were then performed to specifically identify HPV 16, 18, 31, 33 and 35. RESULTS AND CONCLUSIONS: For these high-risk subtypes, LAMP had an overall sensitivity of 99.4% and specificity of 93.2% relative to PCR. LAMP is a promising technology that can accurately diagnose high-risk HPV infection.

Detection of Hepatitis B Virus (HBV) Genomes and HBV Drug Resistant Variants by Deep Sequencing Analysis of HBV Genomes in Immune Cell Subsets of HBV Mono-Infected and/or Human Immunodeficiency Virus Type-1 (HIV-1) and HBV Co-Infected Individuals.

UNLABELLED: The hepatitis B virus (HBV) and the human immunodeficiency virus type 1 (HIV-1) can infect cells of the lymphatic system. It is unknown whether HIV-1 co-infection impacts infection of peripheral blood mononuclear cell (PBMC) subsets by the HBV. AIMS: To compare the detection of HBV genomes and HBV sequences in unsorted PBMCs and subsets (i.e., CD4+ T, CD8+ T, CD14+ monocytes, CD19+ B, CD56+ NK cells) in HBV mono-infected vs. HBV/HIV-1 co-infected individuals. METHODS: Total PBMC and subsets isolated from 14 HBV mono-infected (4/14 before and after anti-HBV therapy) and 6 HBV/HIV-1 co-infected individuals (5/6 consistently on dual active anti-HBV/HIV therapy) were tested for HBV genomes, including replication indicative HBV covalently closed circular (ccc)-DNA, by nested PCR/nucleic hybridization and/or quantitative PCR. In CD4+, and/or CD56+ subsets from two HBV monoinfected cases, the HBV polymerase/overlapping surface region was analyzed by next generation sequencing. RESULTS: All analyzed whole PBMC from HBV monoinfected and HBV/HIV coinfected individuals were HBV genome positive. Similarly, HBV DNA was detected in all target PBMC subsets regardless of antiviral therapy, but was absent from the CD4+ T cell subset from all HBV/HIV-1 positive cases (P<0.04). In the CD4+ and CD56+ subset of 2 HBV monoinfected cases on tenofovir therapy, mutations at residues associated with drug resistance and/or immune escape (i.e., G145R) were detected in a minor percentage of the population. SUMMARY: HBV genomes and drug resistant variants were detectable in PBMC subsets from HBV mono-infected individuals. The HBV replicates in PBMC subsets of HBV/HIV-1 patients except the CD4+ T cell subpopulation.

Hepatitis B virus (HBV) variants fluctuate in paired plasma and peripheral blood mononuclear cells among patient cohorts during different chronic hepatitis B (CHB) disease phases.

Hepatitis B virus is classically considered a hepatotropic virus but also infects peripheral blood mononuclear cells. Chronic hepatitis B has different disease phases modulated by host immunity. We compared HBV variability, drug resistance and immune escape mutations in the overlapping HBV polymerase/surface gene in plasma and peripheral blood mononuclear cells in different disease phases. Plasma and peripheral blood mononuclear cells were isolated from 22 treatment naïve patient cohorts (five inactive, six immune-active, nine HBeAg negative and two immune-tolerant). HBV was genotyped via line probe assay, hepatitis B surface antigen titres were determined by an in-house immunoassay, and HBV DNA was quantified by kinetic PCR. The HBV polymerase/surface region, including full genome in some, was PCR-amplified and cloned, and ~20 clones/sample were sequenced. The sequences were subjected to various mutational and phylogenetic analyses. Clonal sequencing showed that only three of 22 patients had identical HBV genotype profiles in both sites. In immune-active chronic hepatitis B, viral diversity in plasma was higher compared with peripheral blood mononuclear cells. Mutations at residues, in a minority of clones, associated with drug resistance, and/or immune escape were found in both compartments but were more common in plasma. Immune escape mutations were more often observed in the peripheral blood mononuclear cells of immune-active CHB carriers, compared with other disease phases. During all CHB disease phases, differences exist between HBV variants found in peripheral blood mononuclear cells and plasma. Moreover, these data indicate that HBV evolution occurs in a compartment and disease phase-specific fashion.

Molecular characterization of intrahepatic and extrahepatic hepatitis B virus (HBV) reservoirs in patients on suppressive antiviral therapy.

The hepatitis B virus (HBV) replicates via an error-prone reverse transcriptase generating potential drug-resistant quasispecies. The degree of HBV variability in liver vs peripheral blood mononuclear cells (PBMC) in patients on long-term suppressive antivirals is unclear. We characterized HBV replication, drug resistance and molecular diversity in patients with plasma HBV DNA undetectable by clinical assays. Explant liver (n=9), PBMC (n=6) and plasma (n=7) from nine such patients undergoing liver transplantation were evaluated for HBV genomes by sensitive PCR/nucleic acid hybridization assay. Cases with HBV DNA in liver and PBMC were tested for covalently closed circular DNA (HBV cccDNA). HBV polymerase (P) amplicons were cloned, sequenced and both P and overlapping surface (S) gene sequences were analysed. HBV DNA was detected in 43% (3/7) of plasma, 100% (9/9) of liver and 83% (5/6) of PBMC samples. HBV cccDNA was detected in all liver and one PBMC sample. Four patients had a clinical diagnosis of resistance. HBV P gene sequencing revealed 100% wild type (wt) in plasma (2/2), 83% wt in PBMC (5/6) but livers of 3/9 (33%) contained wt and 6/9 (66%) carried resistance to lamivudine and/or adefovir. The translated S gene revealed no changes affecting HBV antigenicity. Sequences from livers with antiviral resistant mutants revealed greater interpatient quasispecies diversity. Despite apparent HBV suppression, the liver continues to support HBV replication and extrahepatic HBV can be detected. PBMC may be a sanctuary for wt virus during antiviral therapy, while the liver harbours more drug-resistant viruses. Drug resistance correlates with intrahepatic viral diversity.

Lentivirus infection in the brain induces matrix metalloproteinase expression: role of envelope diversity.

Infection of the brain by lentiviruses, including human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV), causes inflammation and results in neurodegeneration. Molecular diversity within the lentivirus envelope gene has been implicated in the regulation of cell tropism and the host response to infection. Here, we examine the hypothesis that envelope sequence diversity modulates the expression of host molecules implicated in lentivirus-induced brain disease, including matrix metalloproteinases (MMP) and related transcription factors. Infection of primary macrophages by chimeric HIV clones containing brain-derived envelope fragments from patients with HIV-associated dementia (HAD) or nondemented AIDS patients (HIV-ND) showed that MMP-2 and -9 levels in conditioned media were significantly higher for the HAD clones. Similarly, STAT-1 and JAK-1 levels were higher in macrophages infected by HAD clones. Infections of primary feline macrophages by the neurovirulent FIV strain (V(1)CSF), the less neurovirulent strain (Petaluma), and a chimera containing the V(1)CSF envelope in a Petaluma background (FIV-Ch) revealed that MMP-2 and -9 levels were significantly higher in conditioned media from V(1)CSF- and FIV-Ch-infected macrophages, which was associated with increased intracellular STAT-1 and JAK-1 levels. The STAT-1 inhibitor fludarabine significantly reduced MMP-2 expression, but not MMP-9 expression, in FIV-infected macrophages. Analysis of MMP mRNA and protein levels in brain samples from HIV-infected persons or FIV-infected cats showed that MMP-2 and -9 levels were significantly increased in lentivirus-infected brains compared to those of uninfected controls. Elevated MMP expression was accompanied by significant increases in STAT-1 and JAK-1 mRNA and protein levels in the same brain samples. The present findings indicate that two lentiviruses, HIV and FIV, have common mechanisms of MMP-2 and -9 induction, which is modulated in part by envelope sequence diversity and the STAT-1/JAK-1 signaling pathway.

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences.

To generate an extensive set of subgenomic (sg) mRNAs, nidoviruses (arteriviruses and coronaviruses) use a mechanism of discontinuous transcription. During this process, mRNAs are generated that represent the genomic 5' sequence, the so-called leader RNA, fused at specific positions to different 3' regions of the genome. The fusion of the leader to the mRNA bodies occurs at a short, conserved sequence element, the transcription-regulating sequence (TRS), which precedes every transcription unit in the genome and is also present at the 3' end of the leader sequence. Here, we have used site-directed mutagenesis of the infectious cDNA clone of the arterivirus equine arteritis virus to show that sg mRNA synthesis requires a base-pairing interaction between the leader TRS and the complement of a body TRS in the viral negative strand. Mutagenesis of the body TRS of equine arteritis virus RNA7 reduced sg RNA7 transcription severely or abolished it completely. Mutations in the leader TRS dramatically influenced the synthesis of all sg mRNAs. The construction of double mutants in which a mutant leader TRS was combined with the corresponding mutant RNA7 body TRS resulted in the specific restoration of mRNA7 synthesis. The analysis of the mRNA leader-body junctions of a number of mutants with partial transcriptional activity provided support for a mechanism of discontinuous minus-strand transcription that resembles similarity-assisted, copy-choice RNA recombination.

Characterization of an equine arteritis virus replicase mutant defective in subgenomic mRNA synthesis.

Equine arteritis virus (EAV) is a positive-stranded RNA virus that synthesizes a 5'- and 3'-coterminal nested set of six subgenomic mRNAs. These mRNAs all contain a common leader sequence which is derived from the 5' end of the genome. Subgenomic mRNA transcription and genome replication are directed by the viral replicase, which is expressed in the form of two polyproteins and subsequently processed into smaller nonstructural proteins (nsps). During the recent construction of an EAV infectious cDNA clone (pEAV030 [L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-996, 1997]), a mutant cDNA clone (pEAV030F) which carries a single replicase point mutation was obtained. This substitution (Ser-2429-->Pro) is located in the nsp10 subunit and renders the EAV030F virus deficient in subgenomic mRNA synthesis. To obtain more insight into the role of nsp10 in transcription and the nature of the transcriptional defect, we have now analyzed the EAV030F mutant in considerable detail. The Ser-2429-->Pro mutation does not affect the proteolytic processing of the replicase but apparently affects the function of nsp10 in transcription. Furthermore, our study showed that EAV030F still produces subgenomic positive and negative strands, albeit at a very low level. Both subgenomic positive-strand synthesis and negative-strand synthesis are equally affected by the Ser-2429-->Pro mutation, suggesting that nsp10 plays an important role in an early step of EAV mRNA transcription.

Regulation of coronavirus mRNA transcription.

Coronaviruses synthesize a nested set of six to eight subgenomic (sg) mRNAs in infected cells. These mRNAs are produced in different, but constant, molar ratios. It is unclear which factors control the different levels of sg mRNAs. To determine whether the intergenic sequence (IS) involved in sg mRNA synthesis could affect the transcription efficiencies of other ISs and in this way regulate transcription levels, we inserted multiple ISs at different positions into a mouse hepatitis virus defective interfering RNA. Quantitation of the sg RNAs produced by identical ISs in different sequence contexts led to the following conclusions: (i) transcription efficiency depends on the location of the IS in the defective interfering virus genome, (ii) downstream ISs have a negative effect on transcription levels from upstream ISs, and (iii) upstream ISs have little or no effect on downstream ISs. The observation that a downstream IS downregulates the amounts of sg RNA produced by an upstream IS explains why the smaller sg RNAs are, in general, produced in larger quantities than the larger sg RNAs. Our data are consistent with coronavirus transcription models in which ISs attenuate transcription. In these models, larger sg RNAs are synthesized in smaller amounts because they encounter more attenuating ISs during their synthesis.

Regulation of transcription of coronaviruses.

To study factors involved in regulation of transcription of coronaviruses, we constructed defective interfering (DI) RNAs containing sg RNA promoters at multiple positions. Analysis of the amounts of sg DI RNA produced by these DIs resulted in the following observations: (i) a downstream promoter downregulates an upstream promoter; (ii) an upstream promoter has little or no effect on the activity of a downstream promoter. Our data suggest that attenuation of upstream promoter activities by downstream promoter sequences plays an important role in regulating the amounts of sg RNAs produced by coronaviruses. Our observations are in accordance with the models proposed by Konings et al. and Sawicki and Sawicki.

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus.

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.

A microcomputer-based averager.

A disadvantage of commercially available averagers is the time needed for the output routine. This paper describes a microcomputer, programmed as an averager, with additional features to obtain an examination time as short as possible, which indeed is important for clinical application. Through this apparatus, the time needed for a standard ERG and VECP examination could be reduced from 50 to 25 min.

Two disadvantages of a television system as pattern stimulator for evoked potentials.

The use of television systems to generate patterned stimuli for the examination of EPs has disadvantages due to the frame frequency of 50Hz of television sets. Because of this, changes in the pattern last 20 msec, with the result that the variability in EP latencies is larger than in the results obtained by projector systems. Furthermore, 50 Hz noise on the recordings, transmitter through the visual system, may be seen.

Interference of 50 Hz electrical cortical potentials evoked by TV systems.

Application of TV systems for generating patterned stimuli for the examination of the electrical cortical potentials has disadvantages caused by the frame frequency of 50 Hz of TV sets. Apart from a larger variability in latency times of the electrical potentials as compared to the results obtained by projector systems, a 50-Hz noise on the recordings mediated through the visual system may be seen. These disadvantages can be ameliorated by increasing the frame frequency of the TV set.

Cortical potentials evoked by a TV pattern reversal stimulus with varying check sizes and stimulus field.

With the use of television equipment the influence of check size and stimulus field on the pattern evoked cortical potentials was investigated. Maximum responses were found with 20' and 40' checks. The major part of the responses was initiated from a ring between 1.25 degrees and 2.5 degrees eccentricity, though relatively per stimulus area the response became progressively larger the smaller the stimulus field.

Variation in latency times of visually evoked cortical potentials.

Latency times of visually evoked cortical potentials stimulated by reversal of a slow checkerboard pattern are highly dependent on the time needed to accomplish the reversal movement. If, owing to the method, the pattern reversal time is not kept stable, variability of the latency times is unnecessarily high for clinical purposes. This may be the case when television equipment is used.