Antiviral activity of aqueous and ethanol extracts and of an isolated polysaccharide from Agaricus brasiliensis against poliovirus type 1

AIMS: Agaricus brasiliensis (previously named Agaricus blazei ss. Heinem), also known as the sun mushroom is native of Southeast Brazil, and is widely consumed, mainly in the form of tea, due to its nutritional and pharmacological properties. In this study, we tested aqueous (AqE) and ethanol (EtOHE) extracts and an isolated polysaccharide (PLS) from the fruiting body of A. brasiliensis, for antiviral activity against poliovirus type 1 in HEp-2 cells. METHODS AND RESULTS: The evaluation of the time of addition by plaque assay showed that when AqE, PLS and EtOHE were added, just after the virus inoculation (time 0 h), there was a concentration-dependent reduction in the number of plaques up to 50%, 67% and 88%, respectively, with a selectivity index (SI) of 5.4, 9.9 and 12.3, respectively. CONCLUSIONS: The test substances showed antiviral activity and were more effective when added during the poliovirus infection. As they had little effect on reducing viral adsorption and did not show any virucidal effect, we suggest that they act at the initial stage of the replication of poliovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results corroborate that basidiomycetes can be a rich source of potential antiviral compounds.     

Effects of low temperatures on in vitro produced bovine zygotes

The objective of this research was to investigate the effects of cooling on the development of bovine zygotes. One-cell bovine embryos were maintained at 39°C (control), 20°C, 10°C, or 0°C for 5, 10, or 20 minutes, then cultured in vitro for 7 days and the proportion of embryos developing to the compact morula or blastocyst stage compared between different treatments. Duration of exposure time had no effect on development. Development rates to the compact morula or blastocyst stage were 3.9%, 11.4%, 17.4%, and 24.4% for zygotes maintained at 0°C, 10°C, 20°C, and 39°C, respectively, with differences in embryo yield between every treatment (P < 0.05). In a second experiment, bovine pronuclei (karyoplasts) and cytoplasts were cooled at 0°C or maintained at 39°C for 5 minutes. Pronuclear transplantation was then utilized to create 4 types of reconstructed embryos, those with: 1) non-cooled pronuclei and non-cooled cytoplasm, 2) non-cooled pronuclei and cooled cytoplasm, 3) cooled pronuclei and non-cooled cytoplasm, and 4) cooled pronuclei and cooled cytoplasm. The proportion of embryos developing to the blastocyst stage was highest when non-cooled pronuclei were transferred into non-cooled cytoplasm (18.9%), and similar to that of non-cooled, non-manipulated control zygotes (13.2%, P > 0.05). No embryos developed to the blastocyst stage when pronuclei (cooled or non-cooled) were transferred into cooled cytoplasm. However, zygotes with cooled pronuclei transferred into non-cooled cytoplasm yielded 4.5% blastocysts (P < 0.05). More embryos developed to the compact morula or blastocyst stage when non-cooled vs. cooled cytoplasm was utilized, regardless of whether the pronuclei were cooled (P < 0.05). These data demonstrate that pronuclei are more tolerant to low temperature exposure than is ovum cytoplasm. Mol. Reprod. Dev. 47:435–439, 1997. © 1997 Wiley-Liss, Inc.     

Metal complexes of bovine lactoferrin inhibit in vitro replication of herpes simplex virus type 1 and 2

The inhibitory effect of bovine lactoferrin (BLf) saturated with ferric, manganese or zinc ions, on the infection of Vero cells by human herpes simplex virus type 1 (HSV1) and 2 (HSV2) was investigated. Viral infectivity determined by intracellular antigen synthesis and plaque formation was efficiently inhibited by metal saturated lactoferrins in a dose-dependent manner. Effective BLf concentrations which reduced the infection by 50% ranged from 5.2 to 31 mug ml and were far below the cytotoxicity threshold. Fe BLf and Mn BLf exhibited selectivity indexes higher than Zn BLf and apoBLf for both viruses and the effect was mainly directed towards the early steps of infection. The slight viral inhibition shown by the citrate complexes of the different metals could indicate that the antiviral effect was not significantly influenced by Fe , Mn or Zn ions delivered by BLf into the cells. © Rapid Science 1998     

Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells

Summary Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. Within 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed. This work was supported in part by Public Health Service Research Contract FDA 233-74-1035 and by Research Grant AI-08648 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.     

Effects of donor cells on in vitro development of cloned bovine embryos

The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P<0.05), and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes (P>0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.     

Antiviral activity of lactoferrin towards naked viruses

It is well known that lactoferrin (Lf) is a potent inhibitor towards several enveloped and naked viruses, such as rotavirus, enterovirus and adenovirus. Lf is resistant to tryptic digestion and breast-fed infants excrete high levels of faecal Lf, so that its effect on viruses replicating in the gastrointestinal tract is of great interest. In this report, we analysed the mechanism of the antiviral action of this protein in three viral models which, despite representing different genoma and replication strategies, share the ability to infect the gut. Concerning the mechanism of action against rotavirus, Lf from bovine milk (BLf) possesses a dual role, preventing virus attachment to intestinal cells by binding to viral particles, and inhibiting a post adsorption step. The BLf effect towards poliovirus is due to the interference with an early infection step but, when the BLf molecule is saturated with Zn+2 ions, it is also capable of inhibiting viral replication after the viral adsorption phase. The anti-adenovirus action of BLf takes place on virus attachment to cell membranes through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Taken together, these findings provide further evidence that Lf is an excellent candidate in the search of natural agents against viral enteric diseases, as it mainly acts by hindering adsorption and internalisation into cells through specific binding to cell receptors and/or viral particles.     

Towards an understanding of the poliovirus replication complex: the solution structure of the soluble domain of the poliovirus 3A protein

Poliovirus is a positive-strand RNA virus and the prototypical member of the Picornaviridae family. Upon infection, the viral RNA genome is translated from a single open reading frame into a polypeptide which undergoes a series of cleavages to ultimately form four structural and seven non-structural proteins. A replication complex is then formed which replicates the viral genome into negative and positive strands for further translation, replication, and packaging into viral progeny. Poliovirus 3A protein (3A) is a critical component of the viral replication complex and is the putative target of enviroxime, an antiviral drug shown to block viral replication. 3A also inhibits host cell endoplasmic reticulum-to-Golgi apparatus transport, a function which may play a key role in viral evasion from the host immune response. 3A, an 87-residue protein consisting of a soluble N terminus and a hydrophobic C terminus, is formed by the cleavage of the precursor protein 3AB into 3A and 3B (VPg). Although they differ by only 22 residues, the precursor protein 3AB and its cleavage product 3A have distinct functions in viral replication. We have determined the structure of the soluble, N-terminal domain of 3A (3A-N) using NMR spectroscopy. We show that 3A-N exists as a symmetric dimer, and each monomer consists of an alpha-helical hairpin with unstructured, yet functional, N- and C termini. We also show that the 3A-N structure contains a negatively charged surface patch and provides a context for interpreting the biochemical characteristics of a number of previously reported 3A and 3AB mutants.     

The effect of bovine lactoferrin and lactoferricin B on the ability of feline calicivirus (a norovirus surrogate) and poliovirus to infect cell cultures

AIMS: To characterize the effect of bovine lactoferrin and lactoferricin B against feline calicivirus (FCV), a norovirus surrogate and poliovirus (PV), as models for enteric viruses. METHODS AND RESULTS: Crandell-Reese feline kidney (CRFK) cells were used for the propagation of FCV and monkey embryo kidney (MEK) cells for PV. The assays included visual assessment of cell lines for cytopathic effects and determination of the percentage cell death using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] dye reduction assay. Incubation of bovine lactoferrin with CRFK cells either prior to or together with FCV inoculation substantially reduced FCV infection. In contrast, the interference of lactoferrin with the infection of cells with PV was demonstrated only when lactoferrin was present with cell lines and virus for the entire assay period. Using indirect immunofluorescence, lactoferrin was detected on the surface of both CRFK and MEK cells, suggesting that the interference of viral infection may be attributed to lactoferrin binding to the surfaces of susceptible cells, thereby preventing the attachment of the virus particles. Lactoferricin B, a cationic antimicrobial peptide derived from the N-terminal domain of bovine lactoferrin, reduced FCV but not PV infection. CONCLUSION: Lactoferrin was shown to interfere with the infection of cells for both FCV and PV. However, lactoferricin B showed no interference of infection with PV and interference with infection for FCV required the presence of lactoferricin B together with the cell line and virus. SIGNIFICANCE AND IMPACT OF THE STUDY: An in vitro basis is provided for the effects of bovine lactoferrin and lactoferricin B in moderating food-borne infections of enteric viruses.     

The effects of bovine serum albumin, bovine uterine fluid, and heat-treated bovine serum on in vitro mouse embryo development

Two-cell mouse embryos were cultured in Whitten's medium with one of three supplements: bovine serum albumin (WM + BSA), heat-treated bovine serum (WM + HTBS) or bovine uterine fluid (WM + BUF). Protein concentrations for cultures of WM + BSA were 50.2, 100.5, 251.2, 502.5, and 1005.0 mug/ml and for WM + HTBS were 70.4, 105.1, 269.0, 524.5 and 1193.9 mug/ml. Protein concentrations ranged from 56.9 to 739.1 mug/ml for 22 WM + BUF samples. Embryo development in all media was significantly correlated with the log total protein concentration. When compared to WM + BSA, development was not significantly inhibited or stimulated in any WM + BUF cultures or in WM with 70.4, 524.5 and 1193.9 mug/ml HTBS. Development was enhanced in WM with 105.1 and 269.0 mug/ml HTBS (P<0.05). The results suggest that at the protein concentrations used, culture media supplemented with BUF and BSA support similar mouse embryo development. Culture medium supplemented with HTBS supported embryo development more than medium with BSA. Uterine factors in the bovine capable of enhancing or inhibiting early embryo development were not detected.     

Pulsed UV-light inactivation of poliovirus and adenovirus

AIMS: To study the pulsed ultraviolet (UV) inactivation of poliovirus and adenovirus. METHODS AND RESULTS: Viral suspensions of 2 ml volume were exposed to varying numbers of polychromatic light pulses emitted from a xenon flashlamp. Ten pulses produced an approximately 4 log(10) reduction in poliovirus titre, and no infectious poliovirus remained after 25 pulses. With adenovirus, 10 pulses resulted in an approximately 1 log(10) reduction in infectivity. Adenovirus required 100 pulses to produce an approximately 3 log(10) reduction in infectivity, and 200 pulses to produce a greater than 4 log(10) reduction. CONCLUSIONS: Adenovirus was more resistant to pulsed UV treatment than poliovirus although both viruses showed susceptibility to the treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Pulsed UV-light treatment proved successful in the inactivation of poliovirus and adenovirus, and represents an alternative to continuous-wave UV treatment.     

Serological evidence of alpha herpesvirus infection in sooty mangabeys

Contact between sooty mangabeys (SMs) and a pigtailed macaque prompted the serological screening of SMs for evidence of infection with B virus. Serological tests detected SM antibodies that reacted with B virus polypeptides. Additional testing was performed with sera from SMs with no previous contact with macaques. Results from these tests indicated that 56% (33/59) of the SMs had antibodies that reacted with B virus and SA8. SM antibodies also reacted with herpesvirus papio 2 and to a lesser extent with human alpha herpesviruses (HSV-1 and HSV-2). There was an age-related increase in the presence of these antibodies in SMs that was consistent with the serological pattern of reactivity observed in other nonhuman primate species infected with alpha herpesviruses. These data suggest that SMs may be a host for a herpesvirus that is antigenically similar to those viruses present in other Old World nonhuman primates.     

Localization of norovirus and poliovirus in Pacific oysters

Aims:  To examine the uptake and tissue distribution of norovirus (NoV) and poliovirus (PV) experimentally bioaccumulated in feeding Pacific oysters ( Crassostrea gigas ).
Methods and Results:  Pacific oysters were allowed to bioaccumulated either PV or NoV under tidally synchronized feeding conditions in laboratory tanks. Oysters were then either fixed and paraffin wax embedded prior to localizing virus within tissues by immunohistochemistry (IHC), or they were dissected into digestive tract (stomach, intestine and digestive diverticula), gill and labial palp tissues, and the viral load determined by quantitative RT-PCR. Both PV and NoV immunoreactivities were predominantly found in the lumen and within cells of the digestive tract tissues; however, PV was also found within cells of nondigestive tract tissues, and in the gills and labial palp. Quantitative RT-PCR of tissue extracts corroborate the immunohistochemical data in that the major site for virus localization is the gut, but significant amounts of viral RNA were identified in the gills and labial palp.
Conclusions:  The human enteric viruses, PV and NoV, are readily bioaccumulated by feeding Pacific oysters and that some of the virus is internalized within cells of both digestive and nondigestive tissues.
Significance and Impact of the Study:  Oysters that have been virally contaminated even after depuration (cleaning) in uncontaminated seawater could pose a human health risk if consumed.     

Effects of the dietary supplements, activated charcoal and copper chlorophyllin, on urinary excretion of trimethylamine in Japanese trimethylaminuria patients

Trimethylaminuria (TMAU) is a metabolic disorder characterized by the inability to oxidize and convert dietary-derived trimethylamine (TMA) to trimethylamine N-oxide (TMAO). This disorder has been relatively well-documented in European and North American populations, but no reports have appeared regarding patients in Japan. We identified seven Japanese individuals that showed a low metabolic capacity to convert TMA to its odorless metabolite, TMAO. The metabolic capacity, as defined by the concentration of TMAO excreted in the urine divided by TMA concentration plus TMAO concentration, in these seven individuals ranged from 70 to 90%. In contrast, there were no healthy controls examined with less than 95% of the metabolic capacity to convert TMA to TMAO. The intake of dietary charcoal (total 1.5 g charcoal per day for 10 days) reduced the urinary free TMA concentration and increased the concentration of TMAO to normal values during charcoal administration. Copper chlorophyllin (total 180 mg per day for 3 weeks) was also effective at reducing free urinary TMA concentration and increasing TMAO to those of concentrations present in normal individuals. In the TMAU subjects examined, the effects of copper chlorophyllin appeared to last longer (i.e., several weeks) than those observed for activated charcoal. The results suggest that the daily intake of charcoal and/or copper chlorophyllin may be of significant use in improving the quality of life of individuals suffering from TMAU.     

Natural antibody–complement dependent neutralization of bovine herpesvirus 4 by human serum

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are sensitive or even permissive to BoHV-4. These observations led to the hypothesis that cross-species transmission of BoHV-4 could lead to human infections. In the present study, we investigate the sensitivity of BoHV-4 to neutralization by naïve human sera in order to determine if humans exhibit innate anti-viral activities against this virus. Our results demonstrate that human sera from naïve individuals, in contrast to the sera of naïve subjects from various animal species, neutralize BoHV-4 efficiently. A series of complementary experiments were performed to unravel the mechanism(s) of this neutralization. The data obtained in this study demonstrates that human serum neutralizes BoHV-4 in a complement dependent manner activated by natural antibodies raised against the Galα1-3Galβ1-4GlcNAc-R epitope expressed by bovine cells.     

Efficacy of methylenecyclopropane analogs of nucleosides against herpesvirus replication in vitro

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8). Ten of the analogs had activity against at least one of the viruses tested. One compound had moderate activity against HSV-1 and six had activity against VZV. All but one compound was active against HCMV with a mean EC50 of 2.1 +/- 0.6 microM, compared with a mean EC50 of 3.9 +/- 0.8 microM for ganciclovir. Of special interest was the fact that eight of the ten compounds were active against both HHV-6A and HHV-6B with mean EC50 values of 6.0 +/- 5.2 mciroM and <2.4 +/- 1.5 microM, respectively. Only two compounds had activity against EBV, whereas all but one compound was active against HHV-8 with a mean EC50 of 3.1 +/- 1.7 microM. These results indicate that members of this series of methylenecyclopropane analogs are highly active against HCMV, HHV-6, and HHV-8 but are less active against HSV, VZV, and EBV.     

Comparison of the antiviral action of different human interferons against DNA and RNA viruses

Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α ₂ interferon produced by Escherichia coli containing the human gene for interferon α ₂. All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).     

Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase–polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.     

Production of bovine tetrapolid embryos by electrofusion and their developmental capability in vitro

Optimal conditions of electrofusion for blastomeres of two-cell bovine embryos to produce tetraploid embryos were investigated. The high fusion rate (73–95%), viability, and develop mental capacity were obtained under a field strength of 1.0 kV/cm with direct current pulses of 10 or 25 μsec duration applied twice. Cytological study showed that 78.6% (11/14 embryos) of embryos exposed to electrofusion had tetraploid chromosome sets and the others were diploid or hexaploid. The tetraploid embryos had the capability to develop up to morulae stage in vitro.     

Purification and properties of a host cell protein required for poliovirus replication in vitro

A host cell protein required for poliovirus RNA-dependent RNA replicase activity in vitro has been purified several thousand-fold from an uninfected HeLa cell postmitochondrial supernatant. A single protein of apparent Mr = approximately 67,000 daltons and pI 6.3 is associated with this "host factor" activity. Poly(U)-Sepharose chromatography of the template-dependent replicase isolated from poliovirus-infected cells results in the complete loss of replicase activity if a salt gradient is used to develop the column. Host factor elutes early in the salt gradient and restores replicase activity to protein fractions eluted later in the gradient. The host factor, estimated to be present at 50,000-100,000 copies/cell, interacts physically with replicase.     

Solar disinfection of poliovirus and Acanthamoeba polyphaga cysts in water - a laboratory study using simulated sunlight

AIMS: To determine the efficacy of solar disinfection (SODIS) in disinfecting water contaminated with poliovirus and Acanthamoeba polyphaga cysts. METHODS AND RESULTS: Organisms were subjected to a simulated global solar irradiance of 850 Wm(-2) in water temperatures between 25 and 55 degrees C. SODIS at 25 degrees C totally inactivated poliovirus after 6-h exposure (reduction of 4.4 log units). No SODIS-induced reduction in A. polyphaga cyst viability was observed for sample temperatures below 45 degrees C. Total cyst inactivation was only observed after 6-h SODIS exposure at 50 degrees C (3.6 log unit reduction) and after 4 h at 55 degrees C (3.3 log unit reduction). CONCLUSIONS: SODIS is an effective means of disinfecting water contaminated with poliovirus and A. polyphaga cysts, provided water temperatures of 50-55 degrees C are attained in the latter case. SIGNIFICANCE AND IMPACT OF THE STUDY: This research presents the first SODIS inactivation curve for poliovirus and provides further evidence that batch SODIS provides effective protection against waterborne protozoan cysts.