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.

Characterization of herpes simplex virus clinical isolate Y3369 as a glycoprotein G variant and its bearing on virus typing.

BACKGROUND: Herpes simplex viruses exist as two major serotypes, type 1 (HSV-1) and type 2 (HSV-2). Determination of type, either HSV-1 or HSV-2, is important in accurate diagnosis and clinical control of transmission. Several tests are available for typing HSV, including a monoclonal antibody specific for glycoprotein G and several PCR assays. FINDINGS: A clinical isolate was identified as herpes simplex virus, but tested negative for both HSV-1 and HSV-2 antigens using type-specific monoclonal antibody assays. The isolate was determined to be HSV-1 by PCR analysis. A mutation which likely caused the monoclonal antibody non-reactivity was found in glycoprotein G. Phylogenetic analysis revealed two groups of HSV, one with the mutation and one without. Three population studies examining mutations in HSV-1 glycoprotein G were analyzed by chi-squared test. To this point, the epitope which the monoclonal antibody recognizes was only found in HSV-1 isolates from human European populations (p < 0.0001). CONCLUSIONS: These findings suggest that the PCR-based methods for HSV typing may be more useful than the standard monoclonal antibody test in areas of the world where the variant in glycoprotein G is more prevalent.

Bovine parvovirus uses clathrin-mediated endocytosis for cell entry.

Entry events of bovine parvovirus (BPV) were studied. Transmission electron micrographs of infected cells showed virus particles in cytoplasmic vesicles. Chemical inhibitors that block certain aspects of the cellular machinery were employed to assess viral dependency upon those cellular processes. Chlorpromazine, ammonium chloride, chloroquine and bafilamicin A1 were used to inhibit acidification of endosomes and clathrin-associated endocytosis. Nystatin was used as an inhibitor of the caveolae pathway. Cytochalasin D and ML-7 were used to inhibit actin and myosin functions, respectively. Nocodazole and colchicine were employed to inhibit microtubule activity. Virus entry was assessed by measuring viral transcription using real-time PCR, synthesis of capsid protein and assembly of infectious progeny virus in the presence of inhibitor blockage. The results indicated that BPV entry into embryonic bovine trachael cells utilizes endocytosis in clathrin-coated vesicles, is dependent upon acidification, and appears to be associated with actin and microtubule dependency. Evidence for viral entry through caveolae was not obtained. These findings provide a fuller understanding of the early cell-entry events of the replication cycle for members of the genus Bocavirus.

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 (IC(50)) 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 (CC(50)) 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 IC(50) of 0.025 mg/mL and a CC(50) 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.

Molecular characterization of infectious clones of the minute virus of canines reveals unique features of bocaviruses.

Minute virus of canines (MVC) is a member of the genus Bocavirus in the family Parvoviridae. We have molecularly cloned and sequenced the 5'- and 3'-terminal palindromes of MVC. The MVC genome, 5,404 nucleotides (nt) in length, shared an identity of 52.6% and 52.1% with that of human bocavirus and bovine parvovirus, respectively. It had distinct palindromic hairpins of 183 nt and 198 nt at the left-end and right-end termini of the genome, respectively. The left-end terminus was also found in two alternative orientations (flip or flop). Both termini shared extensive similarities with those of bovine parvovirus. Four full-length molecular clones constructed with different orientations of the left-end terminus proved to be infectious in Walter Reed canine cell/3873D (WRD) canine cells. Both MVC infection and transfection of the infectious clone in WRD cells revealed an identical RNA transcription profile that was similar to that of bovine parvovirus. Mutagenesis of the infectious clone demonstrated that the middle open reading frame encodes the NP1 protein. This protein, unique to the genus Bocavirus, was essential for MVC DNA replication. Moreover, the phospholipase A2 motif in the VP1 unique region was also critical for MVC infection. Thus, our studies revealed important information about the genus Bocavirus that may eventually help us to clone the human bocavirus and study its pathogenesis.

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.

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

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.

Cell death in bovine parvovirus-infected embryonic bovine tracheal cells is mediated by necrosis rather than apoptosis.

The helper-independent bovine parvovirus (BPV) was studied to determine its effect on host embryonic bovine tracheal (EBTr) cells: whether the ultimate outcome of infection results in apoptotic cell death or cell death by necrosis. Infected cells were observed for changes marking apoptosis. Observations of alterations in nuclear morphology, membrane changes, apoptotic body formation, membrane phosphatidylserine inversions, caspase activation and cell DNA laddering in infected cells were not indicative of apoptosis. On the other hand, at the end of the virus replication cycle, infected cells released viral haemagglutinin and infectious virus particles, as would be expected from cell membrane failure. Moreover, the infected cells released lactate dehydrogenase (LDH), release of which is a marker of necrosis. LDH release into the cell medium correlated directly with viral m.o.i. and time post-infection. Furthermore, assessment of mitochondrial dehydrogenase activity was consistent with cell death by necrosis. Taken together, these findings indicate that cell death in BPV-infected EBTr cells is due to necrosis, as defined by infected-cell membrane failure and release of the cell contents into the extracellular environment.

Identification of syncytial mutations in a clinical isolate of herpes simplex virus 2.

Small polykaryocytes resulting from cell fusion are found in herpes simplex virus (HSV) lesions in patients, but their significance for viral spread and pathogenesis is unclear. Although syncytial variants causing extensive fusion in tissue culture can be readily isolated from laboratory strains, they are rarely found in clinical isolates, suggesting that extensive cell fusion may be deleterious in vivo. Syncytial mutations have previously been identified for several laboratory strains, but not for clinical isolates of HSV type 2. To address this deficiency, we studied a recent syncytial clinical isolate, finding it to be a mixture of two syncytial and one nonsyncytial strain. The two syncytial strains have novel mutations in glycoprotein B, and in vitro cell fusion assays confirmed that they are responsible for syncytium formation. This panel of clinical strains may be ideal for examining the effect of increased cell fusion on pathogenesis.

Attachment of bovine parvovirus to sialic acids on bovine cell membranes.

Although it has previously been shown that bovine parvovirus (BPV) attaches to the sialated glycoprotein glycophorin A on erythrocytes, the nature of virus-binding moieties on mammalian nucleated cells is less clear. Buffalo lung fibroblasts (Bu), primary bovine embryonic kidney cells, Madin-Darby bovine kidney cells and bovine embryonic trachea (EBTr) cells were assessed for molecules capable of binding BPV. Competition studies were carried out on both erythrocyte and nucleated cell targets using a variety of sialated compounds and sialic acid-negative compounds. Glycophorin A was found to inhibit BPV binding, while mucin exhibited low-level inhibition. These two sialated compounds also blocked attachment of BPV-modified microsphere carriers to the Bu cell membrane. Influenza A virus was used as a sialic acid competitor and interfered with BPV attachment to erythrocytes and replication in Bu cells. Significantly, the enzyme sialidase removed BPV-binding sites from Bu and EBTr cells. The binding sites could be reconstituted on sialidase-treated cells by the enzymes alpha-2,3-O-sialyltransferase and alpha-2,3-N-sialyltransferase. These results indicated that BPV can attach to sialic acid on cell membranes and that the sialylglycoproteins available for virus attachment appear to contain both N- and O-linked carbohydrate moieties, but that not all members of the sialic acid family can bind BPV. Moreover, there may be other moieties that can bind BPV, which may act as either primary or secondary receptors.

Recovery of an unusual fusogenic herpes simplex virus type 2 strain from a clinical specimen.

A highly unusual herpes simplex virus type 2 strain, strain Burr, was isolated from a female genital tract clinical specimen. This virus induced remarkably rapid and extensive syncytium formation in Vero cells involving hundreds of cells but was less fusion active in HEp-2 cells, MRC-5 cells, and mink lung cells. Virus-infected cells produced the glycoproteins gB, gC, gD and gE.