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Enteroviruses are responsible for emerging diseases which cause diverse symptoms and may result in neurological complications. An antiviral with multiple mechanisms of action can help prevent enterovirus mediated disease despite differences in the pathogenesis between enteroviruses, including the recently identified enterovirus 69 (EV-69) for which pathogenesis is not well understood. This study investigated the efficacy of epigallocatechin-3-gallate stearate (EGCG-S), a modified form of the antioxidant green tea catechin epigallocatechin-3-gallate (EGCG), in inhibiting EV-69 infection of lung fibroblast cells in vitro. Treatment with EGCG-S resulted in moderate protection from EV-69 mediated cytotoxicity as demonstrated by increased metabolic activity as well as maintenance of cell morphology and mitochondrial function. These effects were correlated with reduced hydrogen peroxide production in infected cells following EGCG-S treatment with concentrations less than 100 μM, suggesting a role for inhibition of EV-69 mediated oxidative stress. This study provides insight into characteristics of EV-69 infection as well as the efficacy of EGCG-S mediated inhibition of EV-69 infection.

Epigallocatechin gallate (EGCG), a green tea polyphenol possesses antioxidant, antibacterial, anticancer and antiviral properties. EGCG-Stearate (EGCG-S) is of interest for this study because of its stability and lipophilic properties. The chemical modification of EGCG-S increased its lipid solubility. Herpes simplex virus-1 (HSV-1), a member of the family Herpesviridae, and Alphaherpesvirinae subfamily is a leading cause of human viral diseases in the United States. In this study, 25 μM, 50 μM, 75 μM, and 100 μM of EGCG and EGCG-S were used to carry out cytotoxicity, cell viability and cell proliferation assays to determine the maximum non-cytotoxic concentrations on cultured A549 cells. The results suggested that 75 μM of EGCG and EGCG-S is the appropriate concentration to further study the effect on the infection of HSV-1 in A549 cells. Infectivity, antiviral, and inverted microscopy assays were performed to study the effects of EGCG and EGCG-S on HSV-1 infection. An antiviral assay was performed using luminescence and it indicated that EGCG-S treated HSV-1 showed up to 90% inhibition. Confocal microscopy images further supported the inhibitory effects of 75 μM EGCG-S on HSV-1 infection in A549 cells. The long-term goal of this research is to use EGCG-S as a possible novel topical therapeutic treatment to limit the spread of HSV-1 infections.

The purpose of this study was to investigate the effect of curcumin-treated Herpes simplex virus-1 (HSV-1) and Herpes simplex virus-2 (HSV-2) virions in cultured Vero cells. Previous studies have indicated that curcumin, a polyphenol extracted from the plant Curcuma longa, has demonstrated antiviral properties against a variety of viruses. After establishing the maximum non-cytotoxic concentrations of curcumin on Vero cells, HSV-1 and HSV-2 virions were treated with varying concentrations of curcumin. The effect on infectivity was determined by antiviral assays, using WST-1, plaque assays, adsorption and penetration assays. Treating HSV-1 and HSV-2 viruses with curcumin, at a concentration of 30 μM, reduces the production of infectious HSV-1 and HSV-2 virions in cultured Vero cells by interfering with the adsorption process. These results support the potential of curcumin to be used as a therapeutic agent to reduce the transmission of HSV-1 and HSV-2.

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae, Myoviridae, and Podoviridae) are represented.

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae , Myoviridae , and Podoviridae ) are represented.

Green tea polyphenol epigallocatechin gallate (EGCG) is a strong anti-oxidant that has previously been shown to reduce the number of plaques in HIV-infected cultured cells. Modified EGCG palmitoyl-EGCG (p-EGCG), is of interest as a topical antiviral agent for Herpes Simplex Virus (HSV-1) infections. This study evaluated the effect of p-EGCG on HSV-infected Vero cells. Results of cell viability and cell proliferation assays indicate that p-EGCG is not toxic to cultured Vero cells and show that modification of the green tea polyphenol epigallocatechin gallate (EGCG) with palmitate increases the effectiveness of EGCG as an antiviral agent. Furthermore, p-EGCG is a more potent inhibitor of Herpes Simplex Virus 1 (HSV-1) than EGCG and can be topically applied to skin, one of the primary tissues infected by HSV. Viral binding assay, plaque forming assay, PCR, real-time PCR, and fluorescence microscopy were used to demonstrate that p-EGCG concentrations of 50 µM and higher block the production of infectious HSV-1 particles. p-EGCG was found to inhibit HSV-1 adsorption to Vero cells. Thus, p-EGCG may provide a novel treatment for HSV-1 infections.

To investigate whether rubella virus (RUB) undergoes intermolecular RNA–RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5′-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3′-proximal structural protein (SP) ORF, and maintained the 3′ end of the genome, including the putative 3′ cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3′ half of the genome including the SP-ORF and 3′ CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA–RNA recombination. Using transcripts tagged with a 3′-terminal deletion, it was found that recombinants contained the 3′ end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3′ CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3′ CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3′ end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating selection pressure against duplications in other regions of the genome.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis, kills over 1 million people annually worldwide. With the development of multiple drug resistant strains of TB, interest has increa...

SVlm21 is a mutant of Sindbis virus which was isolated by serial passage of virus in mosquito cells maintained in low-methionine medium; it therefore has a low-methionine-resistant (LMR) phenotype. This phenotype requires mutations at nts 319 and 321; these mutations result in Arg to Leu and Ser to Cys changes at positions 87 and 88 respectively in the viral methyl transferase, nsP1. To better understand the genesis of SVlm21, we carried out serial passages of viruses having only one of these amino acid changes, but in mosquito cells maintained in normal methionine-medium. Whether the passage was begun with SV319 or with SV321, the dominant virus population which emerged always acquired the second SVlm21 amino acid change. However, when the passage was begun with virus having neither the nt 319 or the nt321 mutation, even after many passages neither of these mutations was seen in the passaged virus population. Virus with the LMR phenotype emerged earlier when the virus encoded a wild-type RDRP (passage 4) rather than the mutant RDRP encoded by SVpzf (passage 7). When the methionine concentration in the medium of mosquito cells was increased to 250 µM, more than 20 passages were required until the LMR phenotype predominated. Competition experiments were carried out to compare the relative fitness of SVlm21, SVwt, SV319 and SV321 to each other. Our results indicated that SVlm21 was dominant to SVwt, as well as to both SV319 and SV321. However, SV319 and SV321 were able to co-exist with SVwt implying that in these mixed infection the presence of SVwt inhibited the emergence of SVlm21. Finally, our experiments highlight how a virus population by mutation and selection can adapt to the intracellular concentration of a simple metabolite, S-adenosylmethionine.

Background Sindbis virus (SV) is the prototype of alphaviruses which are a group of widely distributed human and animal pathogens. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein that shuttles between the nucleus and the cytoplasm. Our recent studies found that hnRNP A1 relocates from nucleus to cytoplasm in Sindbis virus (SV)-infected cells. hnRNP A1 binds to the 5' UTR of SV RNA and facilitates the viral RNA replication and translation. Methods Making use of standard molecular techniques, virology methods and an in vitro system developed by our lab to assess the role of hnRNP A1 in SV positive strand RNA synthesis. Results hnRNP A1 interacted with the genomic (G) and subgenomic (SG) RNA promoters. Knockdown of hnRNP A1 resulted in markedly decrease in the synthesis of G and SG RNA both in infected cells and in vitro. Conclusions Our study provides the first direct evidence that hnRNP A1 actively participates in viral RNA replication and is required for the synthesis of G and SG RNA.

Objectives: The purpose of this study was to assess trends in diabetes in pregnancy in American Indian and whites mothers in Montana and North Dakota. Methods: Montana and North Dakota birth records were utilized to assess trends in any diabetes in pregnancy in American Indians and whites from 1989 to 2000. Results: From 1989 through 2000, there were 133,991 and 102,232 births in Montana and North Dakota, respectively. The majority of mothers were American Indian (11%) or white (87%). The rate of any diabetes in pregnancy increased significantly in Montana Indian (3.1–4.1%, p = 0.04) and white mothers (1.8–2.6%, p < 0.001) from 1989–1991 to 1998–2000. The rate also increased significantly in white North Dakota mothers (1.6–3.2%, p < 0.001), but the increase in rate for Indian mothers in North Dakota did not reach statistical significance (3.8–4.8%, p=0.06) during this time period. In each time period, Montana and North Dakota Indian mothers were more likely than white mothers to have any diabetes in pregnancy. Conclusions: The rate of diabetes in pregnancy has increased in American Indian and white mothers. Thus public health programs are now facing an increasing number of women with a history of GDM at future risk of type 2 diabetes and an increasing number of offspring of diabetic pregnancies at risk for becoming overweight and developing type 2 diabetes at a young age.

Background The purpose of this investigation was to determine if black tea extract (BTE), consisting primarily of flavanol compounds called theaflavins, could inhibit herpes simplex virus type-1 (HSV-1) infection in cultured A549 (human epithelial) and Vero cells. Methods The effect of BTE both on A549 and Vero cultured cells and on HSV-1 was assessed by using phase contrast and fluorescent microscopy, and cell viability and proliferation assays. After establishing the maximum non-cytotoxic concentration of BTE, A549 and Vero cells and HSV-1 virions were treated with varying concentrations of BTE, respectively. A549 and Vero cells were infected with HSV-1 with green fluorescent protein (GFP) insert at the UL46 gene. The effect of infectivity was determined by viral DNA extraction followed by PCR, plaque assays, adsorption assays, and electrophoresis of PCR products. Results BTE was not cytotoxic to A549 and Vero cells, as confirmed by cell viability and proliferation assays, in which BTE treated groups paralleled the positive control group. For both cell lines, plaque assays and fluorescent microscopy indicated an inverse relationship between BTE concentration (from 0.14 μM – 1.4 mM) and HSV-1 infectivity. Specifically, PCR and electrophoresis showed a reduction in the viral genome following treatment with BTE. In addition, there was a noticeable decrease in the amount of viral plaques for BTE treated samples in the adsorption assays. Conclusions BTE consisting primarily of theaflavins is not cytotoxic and can reduce or block the production of infectious HSV-1 virions in cultured A549 and Vero cells, thus inhibiting the infectivity of the virus by interfering in the attachment, penetration and viral DNA replication of HSV-1 particles. These findings indicate that BTE enriched with theaflavins has the potential to be developed as a safe, therapeutic antiviral agent to prevent the spread of HSV-1.