F. Brent Johnson

The transcription profile of the Bocavirus bovine parvovirus is unlike those of previously characterized parvoviruses

ABSTRACT The Bocavirus bovine parvovirus generated a single pre-mRNA from a promoter at its left-hand end; however, the pattern of its alternative polyadenylation and splicing was different from that of other parvoviruses. A large left-hand-end open reading frame (ORF) encoded a nonstructural protein of approximately 95 kDa. An abundant, spliced, internally polyadenylated transcript encoded the viral NP1 protein from an ORF in the center of the genome. Transcripts encoding the capsid proteins were polyadenylated in the right-hand terminal palindrome. This is the first published transcription map of a member of the Bocavirus genus of the Parvovirinae.

Comparison of various transport media for viability maintenance of herpes simplex virus, respiratory syncytial virus, and adenovirus

Seven viral transport media (VTM) were compared for effectiveness in preserving the infectivity of herpes simplex virus (HSV), respiratory syncytial virus (RSV), and adenovirus. The media tested were Richards viral transport, sucrose-phosphate-glutamate (SPG), Virocult, HH medium, tryptose phosphate broth, cell culture medium, and Bartels viral transport. Two laboratory strains of HSV (McIntyre and 333) and two clinical isolates (A0301 and A0386), comprising two HSV-1 types and two HSV-2 types, were suspended in each transport medium, kept at 4 degrees C and 22 degrees C, and assayed for surviving virus at various times of up to 2 weeks. Similar testing was done with RSV Long strain and adenovirus type 2. Of the seven media, Richards viral transport, SPG, Virocult, and HH medium, followed closely by tryptose phosphate broth, best preserved HSV infectivity at both 4 degrees C and 22 degrees C. Analysis of decay rates of RSV revealed comparatively rapid decay in SPG and Virocult. Adenovirus was stable in all media and at both temperatures tested. These results indicate that viral transport and subsequent culture isolation are possible using many different VTMs if transport times are kept to a minimum (< 1 day), but if transport extends to longer times, or low levels of virus are present in the specimen, Richards viral transport and HH medium appeared to be the best overall transport media for the viruses tested.

Aminoadamantanes with persistent in vitro efficacy against H1N1 (2009) influenza A.

A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

Virion disruption by ozone-mediated reactive oxygen species

It is well documented in the scientific literature that ozone-oxygen mixtures inactivate microorganisms including bacteria, fungi and viruses (Hoff, J.C., 1986. Inactivation of microbial agents by chemical disinfectants. EPA 600 S2-86 067. Office of Water, U.S. Environmental Protection Agency, Washington, DC; Khadre, M.A., Yousef, A.E., Kim, J.-G., 2001. Microbiological aspects of ozone applications in food: a review. J. Food Sci. 66, 1242-1252). In the current study, delivery and absorption of precisely known concentrations of ozone (in liquid media) were used to inactivate virus infectivity. An ozone-oxygen delivery system capable of monitoring and recording ozone concentrations in real time was used to inactivate a series of enveloped and non-enveloped viruses including herpes simplex virus type-1 (HHV-1, strain McIntyre), vesicular stomatitis Indiana virus (VSIV), vaccinia virus (VACV, strain Elstree), adenovirus type-2 (HAdV-2), and the PR8 strain of influenza A virus (FLUAVA/PR/8/34/H1N1; FLUAV). The results of the study showed that ozone exposure reduced viral infectivity by lipid peroxidation and subsequent lipid envelope and protein shell damage. These data suggest that a wide range of virus types can be inactivated in an environment of known ozone exposure.

Molecular similarities among the adenovirus-associated virus polypeptides and evidence for a precursor protein

Abstract The structural polypeptides (VP1, VP2, and VP3) of adenovirus-associated virus (AAV) were tested for biochemical, immunochemical, and biosynthetic relationships. It was found that the three proteins were similar in their content of leucine, lysine, and aspartic acid. They were tested in radioimmunoprecipitation assays and immunocompetition assays which demonstrated high levels of antigenic similarity. There were also unique regions of antigenic nonhomology. Several nonstructural proteins were found in infected cells, one of which had a molecular weight of 120,000 (VPO) and appeared to have a kinetic relationship with VP1, VP2, and VP3, based on pulse-chase experiments. It was suggested that VPO could be the primary AAV gene product and that it is processed to VP1, VP2, VP3, and other nonstructural fragments during the AAV replicative cycle.

Effect of environmental pH on adenovirus-associated virus.

SummaryThe influence of environmental pH on AAV was studied in infectious virus titrations, induction of CF antigen, production of infectious virus, induction of immunofluorescent stainable antigen, and aggregation of the viral particles. The pH of the medium was found to influence the titer of virus stocks in that less virus was registered at acid pHs, giving differences of up to 105 TCID50 in HEK and HEp-2 cells. Less infectious virus was produced in KB cells, and decreased amounts of CF antigen appeared at acid pHs. However, increased levels of detectable intracellular FA antigen appeared at acid pHs. Electron microscopic examination of AAV particles negatively stained at various pHs showed increasingly large aggregates of particles as the pH was lowered. Under the acid conditions studied, the adenovirus helper and cell activities were only slightly suppressed, with the greatest effect due to aggregation of the virus particles.We gratefully acknowledge the technical assistance of W. M. Hess in the ...Summary The influence of environmental pH on AAV was studied in infectious virus titrations, induction of CF antigen, production of infectious virus, induction of immunofluorescent stainable antigen, and aggregation of the viral particles. The pH of the medium was found to influence the titer of virus stocks in that less virus was registered at acid pHs, giving differences of up to 105 TCID50 in HEK and HEp-2 cells. Less infectious virus was produced in KB cells, and decreased amounts of CF antigen appeared at acid pHs. However, increased levels of detectable intracellular FA antigen appeared at acid pHs. Electron microscopic examination of AAV particles negatively stained at various pHs showed increasingly large aggregates of particles as the pH was lowered. Under the acid conditions studied, the adenovirus helper and cell activities were only slightly suppressed, with the greatest effect due to aggregation of the virus particles. We gratefully acknowledge the technical assistance of W. M. Hess in the electron microscopy study. This study was supported by Public Health Service grant AI-11325 from the National Institute of Allergy and Infectious Diseases.

A rapid culture alternative to the shell-vial method for the detection of herpes simplex virus

The rapid test for detection of herpes simplex virus (HSV) in clinical specimens based on infection of cells in suspension (SI test) was compared to the shell-vial culture (SVC) method and conventional culture. Mink lung cells were used throughout the study. Detection of HSV was not significantly different whether using SI or SVC. The sensitivity of SI in detecting HSV, when compared with conventional culture, was 93.0% using 0.1 ml inocula and 98.3% using 0.5 ml inocula. The time to obtain a final result with both SI and SVC was 1 day compared with 1-7 days by conventional culture. The SI method detected both HSV type-1 and HSV type-2 clinical isolates. The SI technique is a simple method for the rapid detection of HSV and can yield diagnostic results with a minimum of technical manipulations.

Structural polypeptides of adenovirus-associated virus top component

Abstract Low density adenovirus-associated virus (AAV) from upper AAV-adenovirus bands on isopycnic CsCl gradients were purified by sedimentation through sucrose gradients and by immunoprecipitation of filtered particles. These particles appeared to be “empty” capsids because of their uptake of negative stain and their lack of isotopically labeled DNA. Analysis of these particles by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels demonstrated three polypeptides. These polypeptides showed identical molecular weights and the same relative concentrations as the structural polypeptides of marker complete AAV virions when compared by this technique. These findings indicate that the apparently “empty” AAV capsid does not lack any of the three polypeptides found in the complete virus particle, suggesting that none of them are core proteins.

Radioiodination of adenovirus-associated virus external structural proteins.

Abstract The three structural polypeptides of adenovirus-associated virus type 3 (AAV-3) were examined to determine their orientation in the viral capsid. Sepharose-bound lactoperoxidase was used to label both dense-band and major-band AAV-3 virions. All three capsid proteins (VP1, VP2, and VP3) were found to be radioiodinated by solid-state lactoperoxidase in both dense-band and major-band virus particles. These findings indicate that the three polypeptides possess sequences that are externally oriented in the virion and that, therefore, no one of the three polypeptides can be considered an exclusively internal core protein.

Activity of acetone and methanol extracts from thirty-one medicinal plant species against herpes simplex virus types 1 and 2

Context: Thirty-one medicinal plant species from Hawaii, Morocco, and the Sonoran Desert, USA have been shown in past studies to be highly inhibitory to pathogenic bacteria, fungi, and certain cancer cell lines. However, none were tested for antiviral activity. Objective: Acetone and methanol extracts from these species were bio-assayed for antiviral activity against herpes simplex virus types 1 and 2, and for cytotoxicity to the Vero C1008 cell line. Materials and methods: Extracts from these species were tested in vitro for antiviral activity using an immunoperoxidase mini-plaque reduction assay to detect viral structural protein synthesis. A 50% inhibitory concentration (IC50) was computed. Sulforhodamine B and neutral red assays were used to qualitatively and quantitatively assess the cytotoxicity of extracts to C1008 cells, and to compute a 50% cytotoxic concentration (CC50) using a dose response curve. Results: Eight of the 31 plant species assayed showed significant antiviral activity against HSV 1 and HSV 2 viruses. The acetone extract of Kalanchoe pinnata Pers. (Crassulaceae) produced an IC50 of 0.025 mg/mL and a CC50 of 1.25 mg/mL yielding a therapeutic index of 50. Additionally, this extract reduced plaque numbers to zero or near zero at a concentration of 0.1 mg/mL when added 30 min before or 30 min after virus infection. Discussion and conclusion: The mechanism of inhibition against HSV 1 and HSV 2 viruses is now being investigated, along with fractionation of the acetone extract in search of the active compound or compounds.