Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms

Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods

Establishment of an AAV Reverse Infection-Based Array

Background: The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited. Principal Findings: We established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vectormediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments. Conclusions/Significance: Our study provides a novel method for establishing a highly efficient gene transduction arra

Seropositivity to Herpes Simplex Virus Antibodies and Risk of Alzheimer's Disease: A Population-Based Cohort Study

International audienceBACKGROUND: Herpes Simplex Virus (HSV) infection has been proposed as a possible risk factor of Alzheimer's Disease (AD) notably because it is neurotropic, ubiquitous in the general population and able to establish lifelong latency in the host. The fact that HSV was present in elderly subjects with AD suggests that the virus could be a co-factor of the disease. We investigated the risk of developing AD in anti-HSV immunoglobulin G (IgG) positive subjects (indicator of a lifelong infection to HSV) and IgM-positive subjects (indicator of primary infection or reactivation of the virus) in a longitudinal population-based cohort of elderly subjects living in the community. METHODS: Cox proportional hazard models were used to study the risk of developing AD according to the presence or not of anti-HSV IgG and IgM antibodies, assessed in the sera of 512 elderly initially free of dementia followed for 14 years. RESULTS: During the follow-up, 77 incident AD cases were diagnosed. Controlled for age, gender, educational level and Apolipoprotein E4 (APOE4) status, IgM-positive subjects showed a significant higher risk of developing AD (HR = 2.55; 95% CI [1.38-4.72]), although no significant increased risk was observed in IgG-positive subjects (HR = 1.67; 95%CI [0.75-3.73]). No modification effect with APOE4 status was found. CONCLUSION: Reactivation of HSV seropositivity is highly correlated with incident AD. HSV chronic infection may therefore be contributive to the progressive brain damage characteristic of AD

Developmental Regulation of Hepatitis B Virus Biosynthesis by Hepatocyte Nuclear Factor 4α

The host cellular factors that promote persistent viral infections in vivo are, in general, poorly understood. Utilizing the hepatitis B virus (HBV) transgenic mouse model of chronic infection, we demonstrate that the nuclear receptor, hepatocyte nuclear factor 4α (HNF4α, NR2A1), is essential for viral biosynthesis in the liver. The dependency of HBV transcription on HNF4α links viral biosynthesis and persistence to a developmentally regulated transcription factor essential for host viability

A Herpesvirus Encoded Deubiquitinase Is a Novel Neuroinvasive Determinant

The neuroinvasive property of several alpha-herpesviruses underlies an uncommon infectious process that includes the establishment of life-long latent infections in sensory neurons of the peripheral nervous system. Several herpesvirus proteins are required for replication and dissemination within the nervous system, indicating that exploiting the nervous system as a niche for productive infection requires a specialized set of functions encoded by the virus. Whether initial entry into the nervous system from peripheral tissues also requires specialized viral functions is not known. Here we show that a conserved deubiquitinase domain embedded within a pseudorabies virus structural protein, pUL36, is essential for initial neural invasion, but is subsequently dispensable for transmission within and between neurons of the mammalian nervous system. These findings indicate that the deubiquitinase contributes to neurovirulence by participating in a previously unrecognized initial step in neuroinvasion

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin

Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin–sepharose beads and successively mixing an HIV-1 solution with fresh beads.Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (Contract DAAD-19-02-D0002)National Institutes of Health (U.S.) (Grant U01-AI074443

Genomic Sequencing and Comparative Analysis of Epstein-Barr Virus Genome Isolated from Primary Nasopharyngeal Carcinoma Biopsy

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, EBV genome contained in a primary NPC tumor biopsy was amplified by Polymerase Chain Reaction (PCR), and sequenced using next-generation (Illumina) and conventional dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Genbank accession number JQ009376) is a type 1 EBV of approximately 171.5 kb. The virus appears to be a uniform strain in line with accepted monoclonal nature of EBV in NPC but is heterogeneous at 172 nucleotide positions. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC patient-derived strains, GD1 and GD2. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels) in comparison to the reference EBV sequence (accession number NC007605). When compared to AG876, a strain derived from Ghanaian Burkitt's lymphoma, we found 322 SNVs, of which 76 were non-synonymous SNVs and were shared amongst the Chinese GD1, GD2 and HKNPC1 isolates. We observed 88 non-synonymous SNVs shared only by HKNPC1 and GD2, the only other NPC tumor-derived strain reported thus far. Non-synonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. Variations in LMP1 and EBNA3B epitopes and mutations in Cp (11404 C>T) and Qp (50134 G>C) found in GD1, GD2 and HKNPC1 could potentially affect CD8+ T cell recognition and latent gene expression pattern in NPC, respectively. In conclusion, we showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance

Use of Saliva for Early Dengue Diagnosis

The importance of laboratory diagnosis of dengue cannot be undermined. In recent years, many dengue diagnostic tools have become available for various stages of the disease, but the one limitation is that they require blood as a specimen for testing. In many incidences, phlebotomy in needle-phobic febrile individuals, especially children, can be challenging, and the tendency to forgo a dengue blood test is high. To circumvent this, we decided to work toward a saliva-based assay (antigen-capture anti-DENV IgA ELISA, ACA-ELISA) that has the necessary sensitivity and specificity to detect dengue early. Overall sensitivity of the ACA-ELISA, when tested on saliva collected from dengue-confirmed patients (EDEN study) at three time points, was 70% in the first 3 days after fever onset and 93% between 4 to 8 days after fever onset. In patients with secondary dengue infections, salivary IgA was detected on the first day of fever onset in all the dengue confirmed patients. This demonstrates the utility of saliva in the ACA-ELISA for early dengue diagnostics. This technique is easy to perform, cost effective, and is especially useful in dengue endemic countries