Limited differences in fish and benthic communities and possible cascading effects inside and outside a protected marine area in Sagres (SW Portugal).

Marine protected areas (MPAs) are a relatively recent fisheries management and conservation tool for conservation of marine ecosystems and serve as experimental grounds to assess trophic cascade effects in areas were fishing is restricted to some extent. A series of descriptive field studies were performed to assess fish and benthic communities between two areas within a newly established MPA in SW Portugal. We characterized benthic macroalgal composition and determined the size, density and biomass of the main benthic predatory and herbivorous fish species as well as the main benthic herbivorous invertebrates to assess indications of top-down control on the phytobenthic assemblages. Fish species were identical inside and outside the MPA, in both cases Sarpa salpa was the most abundant fish herbivore and Diplodus spp. accounted for the great majority of the benthic predators. However, size and biomass of D. spp. were higher inside than outside the MPA. The main herbivorous invertebrate was the sea urchin Paracentrotus lividus, which was smaller and predominantly showing a crevice-dwelling behaviour in the MPA. In addition, P. lividus size frequency distribution showed a unimodal pattern outside and a bimodal pattern inside the MPA. We found significant differences in the algal assemblages between inside and outside the MPA, with higher abundance of turf and foliose algae inside, and articulated calcareous and corticated macrophytes outside the MPA, but no differences in the invasive Asparagopsis spp. The obtained results show differences in predatory fish and benthic community structure, but not in species richness, inside and outside the MPA. We hypothesize these differences lead to variation in species interactions: directly through predation and indirectly via affecting sea urchins behavioural patterns, predators might drive changes in macroalgal assemblages via trophic cascade in the study area. However due to non-biological differences between the two areas it is difficult to suggest that the MPA causes increased biological parameters of targeted species and to assess predatory control and trophic cascade effects in areas where fishing pressure is reduced. It is therefor advisable to design MPAs so that their impacts can be scientifically evaluated in a proper fashion.

Activity of several S-adenosylhomocysteine hydrolase inhibitors against African swine fever virus replication in Vero cells.

Several inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase have been found to selectively suppress the replication of African swine fever virus (ASFV) in Vero cells. Of the compounds tested, 3-deazaneplanocin A proved to be the most potent and selective inhibitor of ASFV replication. Its selectivity index (SI) was 3000. Then followed 9-(trans-2',trans-3'-dihydroxycyclopentyl)-3- deazaadenine (SI = 2500), the 4'beta-vinyl derivative of 9-(trans-2',trans-3'-dihydroxycyclopentyl)adenine (SI = 2000), 6'beta-fluoroaristeromycin (SI = 1250), 4',5'-unsaturated 5'-fluoroadenosine (MDL 28842) and 9-(trans-2',trans-3'-dihydroxycyclopentyl)adenine (SI = 667), 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)adenine and the 4 beta-methyl derivative of 9-(trans-2',trans-3'- dihydroxycyclopentyl)adenine (SI = 400), 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-3-deazaadenine (SI = 200). We postulate that the mechanism of anti-ASFV action of these compounds is based on the inhibition of AdoHcy hydrolase, thus resulting in the accumulation of AdoHcy and suppression of methylation reactions needed for viral mRNA maturation.

Antiviral activity of sulfated polysaccharides against African swine fever virus.

The polyanionic substances lambda and kappa carrageenan, pentosan polysulfate, fucoidan, dextran sulfate and heparin were investigated for their inhibitory effect on the replication of African swine fever virus (ASFV) in vitro. Lambda carrageenan was the most efficacious with a selectivity index, as based on the ratio of the 50% cytotoxic concentration to the 50% antiviral effective concentration, of 120, followed by pentosan polysulfate with 30, kappa carrageenan 13.3 and fucoidan 10. Dextran sulfate and heparin were almost inactive. In general, the substances had low toxicity for Vero cells. The studies with radiolabeled ASF virions suggest that the sulfated polysaccharides inhibit virus adsorption. Inhibition of virus replication was found for all the polysaccharides only when the substances were present during virus adsorption, with the exception of lambda and kappa carrageenan, which were also inhibitory when added immediately after the adsorption period.

Interferon cures cells lytically and persistently infected with African swine fever virus in vitro.

Human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) inhibit African swine fever (ASF) virus replication in Vero cells. IFN-alpha and IFN-gamma exert a synergistic inhibition. Human tumor necrosis factor (TNF) does not inhibit ASF virus replication in this cell line, but in combination with IFNs it has antiviral enhancing activity. Analysis of the mechanism of inhibition suggests that the action of these cytokines blocks a step that comes prior to DNA replication. The 2'-5' A synthetase activity is induced in Vero cells by treatment with these cytokines and is activated after ASF virus infection. More interesting is the finding that continuous treatment with IFN-alpha cures Vero cells from lytic and persistent infections with ASF virus. A potential application of IFN for the treatment of animals carrying the virus is suggested.

Effect of a uridine 5'-diphosphate glucose analogue on herpes simplex keratitis in rabbits and vaginal infection in guinea pigs.

A uridine 5'-diphosphate glucose analogue, active in vitro against herpes simplex type 1 and 2 viruses, was assayed in rabbit infected corneas with the above viruses. The infected eyes were treated by drug instillation thrice daily and evaluation of ocular lesion was performed by slit-lamp biomicroscopy. The compound [[[5'-(2'',3'',4'',6''-tetra-O-benzoyl-alpha-D-glucopyranosyl)oxy] carbonyl]amino)sulfonyl]uridine shows a moderate antiviral activity, resulting in a reduction in the severity of clinical illness during acute infection. Vaginal infection of guinea pigs with herpes simplex type 2 virus was treated topically by instillation twice daily with the compound. The effect on clinical evolution was related to viral shedding from the genital tract, and a moderate reduction of both parameters in respect to the infected untreated controls was observed.

Inhibition of African swine fever virus DNA synthesis by (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine.

The acyclic nucleotide analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] is a potent and selective inhibitor of African swine fever virus (ASFV) replication. Using the DNA-DNA hybridization technique with plasmid pRPEL-2 as probe, we have shown that (S)-HPMPA exerts a specific, dose-dependent, inhibitory effect on viral DNA synthesis. Also, (S)-HPMPA inhibits the production of late viral proteins, especially IP-73, in ASFV-infected MS and Vero cells. When evaluated under the same experimental conditions, phosphonoacetic acid (PAA) also caused an inhibition of viral DNA and late viral protein synthesis but only so at a concentration which was 10- to 20-fold higher than that required for (S)-HPMPA.

A model virus-cell system to study the persistence of African swine fever virus.

The persistence of African swine fever virus (ASFV) on Vero cells was induced by using 5-iodo-2'-deoxyuridine (IDU). After the persistence was established, several cycles of decreasing virus production were observed with intervals in which no virus could be detected. These latency-like periods could last from 15 to 25 days. After three and a half months the cells appeared to be "cured" and no virus was detected during almost three years. These "cured" cells (Vero-L) were more resistant to superinfection with the wild type virus, and when infected they always established persistence without drug addition characterized by a continuous virus production. The persistent virus isolated at passage 23rd from ASFV persistently infected Vero-L cells was different from wild type in a) the morphology of the plaque, b) its ability to replicate in Vero-L cells, and c) greater resistance to be inhibited by IDU in normal Vero cells (Vero-N). These results suggest that both, Vero cells and ASFV have changed during persistent infection.

Phosphonylmethoxyalkylpurines and -pyrimidines as inhibitors of African swine fever virus replication in vitro.

Several phosphonylmethoxyalkylpurine and -pyrimidine derivatives related to (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) were evaluated as inhibitors of African swine fever virus (ASFV) replication in Vero cells. (S)-HPMPA has previously been shown to inhibit ASFV replication at a minimum inhibitory concentration (MIC) of 0.01 microgram/ml with a selectivity index of 15000. Of the new compounds tested, the following emerged as the most potent and selective inhibitors of ASFV replication: the cyclic phosphonate of (S)-HPMPA [(S)-cHPMPA] with an MIC of 0.2 microgram/ml and a selectivity index of 2500, the 2,6-diaminopurine analogue of (S)-HPMPA [(S)-HPMPDAP] with an MIC of 0.5 microgram/ml and a selectivity index of 1400, and the cytosine [(S)-HPMPC] and guanine [(RS)-HPMPG] analogues with an MIC of 1 microgram/ml and a selectivity index of 600-700.

Comparative efficacy of broad-spectrum antiviral agents as inhibitors of African swine fever virus replication in vitro.

Various nucleoside analogues, selected on the basis of their previously established broad-spectrum antiviral properties, were evaluated for their potency and selectivity as inhibitors of the in vitro replication of the iridovirus, African swine fever virus (ASFV). The test compounds included (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)adenine, (RS)-3-adenin-9-yl-2-hydroxypropanoic acid isobutyl ester, (S)-9-(2,3-dihydroxypropyl)adenine, carbocyclic 3-deazaadenosine (C-c3Ado), 3'-azido-2',3'-dideoxythymidine, pyrazofurin and ribavirin. As the most efficacious inhibitors of ASFV replication emerged (S)-HPMPA followed by C-c3Ado. The minimum inhibitory concentration of (S)-HPMPA for ASFV replication was 0.01 microgram/ml, and its selectivity index was 15,000. The corresponding values for C-c3Ado were 0.025 micrograms/ml and 8000, respectively. It would seem justified to further pursue these compounds for their anti ASFV activity in vivo.

New agents active against African swine fever virus.

Actinobolin, atropine, carrageenan, megalomycin C, suramin, and tetracenomycin C were tested for their activity against African swine fever virus replication. Both viral inhibitory potency and cytotoxicity were investigated. Megalomycin C, suramin, atropine, and carrageenan exhibited significant activity. Megalomycin C was the most active of the four agents with respect to the concentration of compound that blocked the formation of infectious virus by 50%. Suramin was the next most active agent in this respect, but because of its lower cytotoxicity, it had the most favorable therapeutic index.

Inactivation and inhibition of African swine fever virus by monoolein, monolinolein, and gamma-linolenyl alcohol. Brief report.

Monoolein, monolinolein and (most strongly) gamma-linolenyl alcohol inactivate ASF virus and inhibit its replication in Vero cells at 25 micrograms/ml while at 10 micrograms/ml no inactivation occurs but inhibition of replication in tissue culture is observed. This suggests two possibly different action mechanisms.

Antiviral action of 5-amino-2-(2-dimethyl-aminoethyl)benzo-[de]-isoquinolin-1,3-dion e.

A newly synthesized imide derivative of 3-nitro-1,8-naphthalic acid, 5-amino-2-(2-dimethylaminoethyl)benzo-[de]-isoquinolin-1,3-dione (designated M-FA-142), was tested on chick embryo cells against herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV), and on Vero cells against African swine fever virus (ASFV). At a concentration of 4 micrograms/ml the drug inhibited VV replication by about one order of magnitude, and that of HSV-1 by about three orders of magnitude. A minor effect was shown against ASFV. Virus inhibition was found to depend on the amount of drug and multiplicity of infection. No virucidal effect was observed on the viruses tested, except for a slight effect on HSV-1. Inhibition of virus growth could be reversed when the drug was removed from the cell culture medium. Serial passages of HSV-1 and VV in the presence of the drug caused the appearance of drug-resistant viruses.

Imide derivatives of 3-nitro-1.8-naphthalic acid: their inhibitory activity against DNA viruses.

The antiviral action of a new drug, 5-amino-2-(dimethylaminoethyl)-benzo-[de]-isoquinolin-1.3-dione has been studied against herpes simplex type 2 (HSV-2) and adenovirus type 5 (Ad-5) grown in Vero cells. The concentration of the drug which gives a 5 log10 reduction in virus titer was 4 micrograms/ml (maximum tolerated concentration) for HSV-2. The anti-HSV-2 activity of this compound was one log. more powerful than that of iododeoxyuridine (IDU). At this concentration the drug shows virucidal activity against HSV-2. No inhibition was found when the drug was tested against Ad-5. The inhibition of virus production has been studied depending upon the amount of drug or virus, and drug addition-time after infection. Reversibility of inhibition after drug removal, and drug resistance in the presence of the drug have also been determined.

Effect of disodium phosphonoacetate and iododeoxyuridine on the multiplication of African swine fever virus in vitro.

Disodium phosphonoacetate (PAA) was found to inhibit the replication of African swine fever virus (ASFV). The action of this compound has been compared with the inhibitory capacity of iododeoxyuridine (IDU) upon ASFV growing in Vero cells. The study was done by the immunofluorescence technique in order to detect formations of cytoplasmic virus antigens and inclusion bodies; both were found to be inhibited by IDU and PAA. At 100 microgram/ml, IDU blocked completely the multiplication of ASFV and with PAA, a few scattered cells showed positive fluorescence. The infectivity of the virus was reduced 1--5 log depending upon drug concentrations and time of exposure to the drugs. Inhibition of ASFV replication by PAA suggests that this virus, like other herpesviruses, involves a virus-specific DNA polymerase in its replication mechanism.

Plaque formation by African swine fever virus in chick embryo fibroblasts in the absence of CO2 atmosphere.

A plaque assay method using ASFV previously adapted to growth in chick embryo fibroblasts is described. Chick embryo fibroblast monolayers under bactoagar or methylcellulose have been employed using cysteine, arginine, DEAE-Dextran and HEPES as additives. Plaque production was optimal under methylcellulose. HEPES rendered the plaques more clear when used with the overlay. Arginine enhances plaque formation with bactoagar, and DEAE-Dextran doubles the plaque size. The growth curve of ASFV in chick embryo monolayers has been studied.