Mangiferin: A promising natural xanthone from Mangifera indica for the control of acyclovir - resistant herpes simplex virus 1 infection.

Mangiferin is found in many plant species as the mango tree (Mangifera indica) with ethnopharmacological applications and scientific evidence. The emergence of resistant herpes simplex virus (HSV) strains to Acyclovir (ACV) has encouraged the search for new drugs. We investigated the in vitro and in vivo activity of mangiferin obtained from M. indica against ACV-resistant HSV-1 (AR-29) and sensitive (KOS) strains. The in vitro activity was performed under varying treatment protocols. The substance showed a CC50 > 500 µg/mL and IC50 of 2.9 µg/mL and 3.5 µg/mL, respectively, for the AR-29 and KOS strains. The in vivo activity was performed in Balb/c mice treated with 0.7% topical mangiferin formulation. This formulation inhibited most effectively the AR-29 strain, attenuated the lesions, postponed their appearance or enhanced healing, in comparison to control group. We demonstrated the potentiality of mangiferin from M. indica to control HSV replication with emphasis to ACV-resistant infection.

Antiherpetic Effect of Topical Formulations Containing Sulfated Polysaccharide from Adenanthera pavonina.

Adenanthera pavonina is a native tree of Africa and Asia, introduced in Brazil for reforestation and wood industry. Several pharmacological activities have described scientifically, including antiviral activity. This study evaluated the antiviral effect of sulfated polysaccharide of Adenanthera pavonina (SPAp) against acyclovir (ACV)-resistant (AR-29) and sensitive (KOS) herpes simplex virus strains. The 50% cytotoxic concentration (CC50) was determined by MTT method and the 50% inhibitory concentration (IC50) was evaluated by plaque reduction assay. The in vivo SPAp antiviral activity was performed in Balb/c mice infected by skin scarification and treated with topical 0.5% (w/w) SPAp formulations. SPAp showed a CC50 of 47.81 µg/mL and the IC50 were 0.49 µg/mL (SI = 97.5) and 0.54 µg/mL (SI = 88.5) for the strains KOS and AR-29, respectively. Our results demonstrated that mice treated with SPAp presented a delay in the development and progression of skin lesions compared with the control group.

Polysaccharide and extracts from Lentinula edodes: structural features and antiviral activity.

BACKGROUND: Lentinula edodes, known as shiitake, has been utilized as food, as well as, in popular medicine, moreover, compounds isolated from its mycelium and fruiting body have shown several therapeutic properties. The aim of this study was to determine the antiviral activity of aqueous (AqE) and ethanol (EtOHE) extracts and polysaccharide (LeP) from Lentinula edodes in the replication of poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BoHV-1). METHODS: The time-of-addition assay was performed at the times -2, -1, 0, 1 and 2 h of the infection. The virucidal activity and the inhibition of viral adsorption were also evaluated. Plaque assay was used to monitor antiviral activity throughout. RESULTS: The AqE and LeP were more effective when added at 0 h of infection, however, EtOHE was more effective at the times 1 h and 2 h of the infection. AqE, EtOHE and LeP showed low virucidal activity, and the inhibition of viral adsorption was not significant. CONCLUSIONS: The results allowed us to conclude that AqE, EtOHE and LeP act on the initial processes of the replication of both strains of virus.

Cucumis melo pectin as potential candidate to control herpes simplex virus infection.

The herpes simplex virus, also known as HSV, is an important human pathogen. Acyclovir (ACV) is the first-line antiviral for the treatment of HSV infections; nevertheless HSV resistance to ACV has been increasingly reported and, therefore, search for alternative drugs have been encouraged. Herein, the effect of Cucumis melo sulfated pectin (SPCm) was evaluated in the HSV-1 infection. Pectin cytotoxicity and its antiherpetic action were determined by assays of MTT and plaque reduction, respectively. The SPCm concentration that reduced the cell viability by 50% (CC50) was 1440 µg/mL, while the concentration that reduced PFU in 50% (IC50) was 6 µg/mL against ACV-sensitive (KOS) strain and 12 µg/mL for ACV-resistant (AR-29) strain. The pectin showed high selectivity index (SI) for both viral strains. Therefore, we suggest that SPCm has been effective for HSV-1, strenghten by viral protein and DNA syntheses inhibition. In conclusion, we have found that SPCm is a promising alternative compound to control HSV infection.

Assessment of antiherpetic activity of nonsulfated and sulfated polysaccharides from Azadirachta indica.

Azadirachta indica leaf is used by Indian population for the healing of various diseases including viral infection. Herein, we analyzed the antiherpetic (HSV-1) activity of two polysaccharides (P1 and P2) isolated from the leaf of A. indica and their chemically sulfated derivatives (P1S and P2S). The molecular weights of P1S and P2S are 41 and 11 kDa, respectively. Sulfate groups are located at positions C3 of the Araf and C6 of both Galp and Glcp residues of the most active polysaccharide (P1S). These compounds were not cytotoxic in HEp-2 cells, up to 1000 µg/mL. Both P1S and P2S exhibited antiviral activity when used simultaneously to HSV-1, with 50% inhibitory concentration/selectivity index, respectively, of 31.1 µg/mL/>51.4 and 80.5 µg/mL/>19.8. P1S showed better inhibitory effect (91.8%) compared to P1 (50%), P2 (71.1%) and P2S (70%) at 200 µg/mL. Synthesis of viral protein showed a dose-dependent response and the nucleic acid synthesis was inhibited up to 25 µg/mL, by P1 and P1S and up to 50 µg/mL, by P2 and P2S. The antiviral effect is probably due to the interference of polysaccharides at the early stages of HSV-1 replication, including adsorption. Further studies are under way to get insight into the mechanism of action of the substances.

Structural characterization and antiviral activity of pectin isolated from Inga spp.

Several studies have been conducted on polysaccharides derived from natural sources, and their different biological properties have been reported. Their low toxicity and antiviral effects i.e., their action on several steps of viral replication, have been extensively examined. In this work, pectin isolated from Inga spp. fruit pulp was first characterized and evaluated using HEp-2 cells against the herpes simplex virus type 1 (HSV-1) and the poliovirus (PV). The isolated pectin (denoted as PDTS) was characterized by infrared spectroscopy, NMR and Gel permeation chromatography. The cytotoxicity was analyzed by the MTT method and antiviral activity by plaque reduction assay, immunofluorescence assay (IF) and polymerase chain reaction (PCR). The cytotoxic concentration (CC50) of PDTS was 870 µg.mL-1 and the inhibitory concentrations (IC50) were 179 µg.mL-1 and 58 µg.mL-1 for HSV-1 and PV, respectively. Greater inhibitory effect was observed when the cells were simultaneously treated with PDTS and infected, suggesting that PDTS inhibited the initial viral replication stages, revealing its antiviral potential.

The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca.

We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal plant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 cells, and the 50% inhibitory concentration (IC50) were 22.01 microg/ml (selectivity index (SI)=2.83) and 21.10 microg/ml (SI=2.95), respectively. At the highest concentration of the drug (40 microg/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains.

Herpesvirus: an underestimated virus.

Herpes simplex virus (HSV) infections are common and widespread; nevertheless, their outcome can be of unpredictable prognosis in neonates and in immunosuppressed patients. Anti-HSV therapy is effective, but the emergence of drug-resistant strains or the drug toxicity that hamper the treatment is of great concern. Vaccine has not yet shown relevant benefit; therefore, palliative prophylactic measures have been adopted to prevent diseases. This short review proposes to present concisely the history of HSV, its taxonomy, physical structure, and replication and to explore the pathogenesis of the infection, clinical manifestations, laboratory diagnosis, treatment, prophylaxis and epidemiology of the diseases.

Green seaweed Enteromorpha compressa (Chlorophyta, Ulvaceae) derived sulphated polysaccharides inhibit herpes simplex virus.

The herpes simplex virus (HSV) diseases represent a relevant medical and social problem due to their communicability and recurrence following latency. The green algae are rich source of polysaccharides referred to as ulvans, reported as being biologically and pharmacologically active. In this work, we analyzed the activity of seven chemically modified polysaccharides from Enteromorpha compressa (Chlorophyta, Ulvaceae), against HSV. Only the derivative named SU1F1 showed satisfactory viral inhibition activity, with a high selectivity index, and, therefore, it was submitted to analysis of the probable mechanism of action and structure. SU1F1 is a sulphated (22% w/w) heteroglycuronan with an apparent molecular mass of 34kDa. The antiviral activity was assayed by plaque reduction assay under the protocols of the time-of-addition (from 3h before infection to 16h after infection), the inhibition of virus adsorption and penetration, and the virucidal effects. SU1F1 showed a high viral activity at the time 0h. We demonstrated that its inhibitory effect was maintained until 4h post-treatment with 100% of viral inhibition at 100µg/ml. No effect was observed in additional protocols (the pre-treatment, the inhibition of adsorption and penetration and virucidal assays). Reverse Transcriptase associated PCR (RT-PCR) results were in accordance with plaque reduction assay and demonstrated the activity of SU1F1 at the initial stages of HSV replication.

Antiviral effect of Guazuma ulmifolia and Stryphnodendron adstringens on poliovirus and bovine herpesvirus.

Crude extract (CE) and aqueous (AqF) and ethyl acetate (EtOAcF) fractions of Guazuma ulmifolia Lam., Sterculiaceae and the corresponding AqF, EtOAcF of Stryphnodendron adstringens (Mart.) Coville, Leguminosae were tested for their antiviral activity against poliovirus 1 (P-1) and bovine herpesvirus 1 (BHV-1) in HEp-2 cultured cells. The antiviral activity was monitored by plaque assay and immunofluorescence assay (IFA) under virucidal and therapeutic protocols. The therapeutic protocol demonstrated statistically significant positive results with both plants and for both virus strains. The highest percentages of viral inhibition were found for G. ulmifolia EtOAcF which inhibited BHV-1 and P-1 replication by 100% and 99%, respectively (p<0.05, Student's t-test). For S. adstringens, AqF was the most efficient, inhibiting BHV-1 and P-1 by 97% and 93%, respectively (p<0.05). In the virucidal protocol, G. ulmifolia CE inhibited the replication of BHV-1 and P-1 by 60% and 26%, respectively (p<0.05), while, for S. adstringens, inhibition of 62% (p<0.05) was demonstrated only with EtOAcF for P-1. IFA demonstrated that the greatest reduction in fluorescent cell number occurred with G. ulmifolia, under the therapeutic protocol for both virus strains. However, AqF and EtOAcF of S. adstringens were most efficient with the virucidal protocol for P-1. In conclusion, we demonstrated that G. ulmifolia and S. adstringens inhibited BHV-1 and P-1 replication, as well as, blocked the synthesis of viral antigens in infected cell cultures.

Use of acridine orange staining for the detection of rotavirus RNA in polyacrylamide gels.

Acridine orange is a metachromatic intercalator used extensively in histochemistry to differentiate double- from single-stranded (ds, ss) nucleic acid by the emission of green and red fluorescence, respectively, under ultraviolet light. In the present study we standardised a protocol in order to use acridine orange to detect rotavirus ds RNA in polyacrylamide gels and compared it to silver and ethidium bromide staining. We demonstrated that the simplest and best condition was attained when gels containing rotavirus ds RNA bands, stained in green, were treated with 4.3 microM acridine orange after electrophoresis and destained with distilled water pH 6 at 37 degrees C. Under this protocol, rotavirus RNA concentration was calculated and the mean minimum amounts of nucleic acid detected by acridine orange, ethidium bromide, and silver staining were 26.0 +/- 4.29, 15.6 +/- 1.48 and 1.06 +/- 0.11 ng, respectively. The comparison of acridine orange sensitivity with ethidium bromide and silver staining, for 25 field strains of rotavirus and one cell-adapted strain (SA11), demonstrated concurrent results in 80% of the specimens. Red colour emission resulting from the interaction of acridine orange with ss nucleic acid was also shown by testing denatured 0.5 kb HindIII digest of lambda phage DNA. Furthermore, it was demonstrated that rotavirus ds RNA could be used for reverse transcription activity, followed by PCR amplification, after acridine orange staining. In conclusion, although acridine orange is less sensitive than ethidium bromide and silver staining, its practicality, low cost, metachromatic properties, and its non-interference on RT-PCR should be considered. It is suggested the use of acridine orange as an appropriate stain for various purposes in virology, as well as for the molecular biology of nucleic acid.

Antiviral Activity of Sulfated Polysaccharide of Adenanthera pavonina against Poliovirus in HEp-2 Cells.

Adenanthera pavonina, popularly known as red-bead tree, carolina, pigeon's eye, and dragon's eye, is a plant traditionally used in Brazil for the treatment of several diseases. The present study aimed at evaluating the activity of sulfated polysaccharide from the Adenanthera pavonina (SPLSAp) seeds against poliovirus type 1 (PV-1) in HEp-2 cell cultures. The SPLSAp presented a cytotoxic concentration (CC50) of 500 µg/mL in HEp-2 cell cultures, evaluated by the dimethylthiazolyl-diphenyltetrazolium bromide method (MTT). The SPLSAp exhibited a significant antiviral activity, with a 50% inhibitory concentration (IC50) of 1.18 µg/mL, determined by plaque reduction assay and a high selectivity index (SI) of 423. The maximum inhibition (100%) of PV replication was found when the SPLSAp treatment was concomitant with viral infection (time 0 h), at all tested concentrations. The maximal inhibition was also found when the SPLSAp was used 1 h and 2 h postinfection, albeit at 50 µg/mL and 100 µg/mL. Therefore, we demonstrated that the SPLSAp inhibited PV growth. We also suggested that SPLSAp inhibited PV in more than one step of the replication, as the mechanism of antiviral action. We, therefore, selected the compound as a potential candidate for further development towards the control of the infection.

Herpes simplex virus: isolation, cytopathological characterization and antiviral sensitivity.

BACKGROUND: Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually self-limiting, though they can be serious, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resistant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensitivity are important in the establishment of an antivirogram for controlling the infection. OBJECTIVE: This study sought to isolate and examine the cytopathological characteristics of circulating strains of the Herpes simplex virus, from clinical specimens and their sensitivity to commercially available antiherpesvirus drugs, acyclovir, phosphonophormic acid and trifluridine. METHODS: Herpes simplex virus isolation, cytopathological features and antiviral sensitivity assays were performed in cell culture by tissue culture infectious dose or plaque forming unit assay. RESULTS: From twenty-two clinical specimens, we isolated and adapted nine strains. Overall, the cytopathic effect was detected 24 h post-infection (p.i.) and the presence of syncytia was remarkable 48 h p.i., observed after cell staining. Out of eight isolates, four developed plaques of varying sizes. All the isolates were sensitive to acyclovir, phosphonophormic and trifluridine, with the percentage of virus inhibition (%VI) ranging from 49.7-100%. CONCLUSIONS: The methodology for HSV isolation and characterization is a straightforward approach, but the drug sensitivity test, regarded as being of great practical importance, needs to be better understood.

Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and poliovirus.

Herpes simplex virus (HSV) is one of the most regular human pathogens, being a public health problem, and causal agent of several diseases. Poliovirus (PV) is an enteric virus and about 1% of infected individuals develop paralytic poliomyelitis due to viral invasion of the central nervous system and destruction of motor neurons. This work evaluated the activity of a sulfated polysaccharide of Caesalpinia ferrea (SPLCf) in HSV and PV replication. The antiviral effect of SPLCf at varying concentrations was tested by plaque assay under several protocols, such as time-of-addition, adsorption and penetration inhibition and virucidal. Syntheses of viral protein and nucleic acid were also monitored by the immunofluorescence assay and PCR. The SPLCf inhibited virus adsorption and steps after penetration, and inhibited the synthesis of viral protein. Virucidal effect was also shown and nucleic acid synthesis was concurrent with positive results. Our findings suggested that the substance with low toxicity represent a potential viral inhibitor.

Antiherpetic activity of an Agaricus brasiliensis polysaccharide, its sulfated derivative and fractions.

Agaricus brasiliensis is an edible mushroom, traditionally used for the treatment of several diseases. In this paper, a polysaccharide (PLS) from A. brasiliensis, its carboxymethylated (CPLS) and sulfated (SPLS) derivatives, as well as, fractions (F1-F3) obtained from the PLS were investigated for their effect in the replication of herpes simplex virus and bovine herpes virus in HEp-2 cell cultures. The PLS, SPLS and F3 inhibited both virus strains similarly, in a dose-dependent curve. F1, F2 and CPLS did not show significant effect even at higher concentrations. All the compounds showed neither virucidal or viral adsorption inhibition activities nor effect when cells were treated prior to infection. Our study demonstrated that the extracts of A. brasiliensis, can be promising for future antiviral drug design and its biotechnological production is economically feasible.

Detection and characterization of human rotavirus in hospitalized patients in the cities of Ponta Grossa, Londrina and Assai - PR, Brazil.

UNLABELLED: Acute diarrheal disease is still one of the major public health problems worldwide. Rotaviruses (RV) are the most important viral etiologic agents and children under five years of age are the target population. OBJECTIVE: To investigate the rate of RV infection in hospitalized patients due to acute diarrhea in the cities of Ponta Grossa, Londrina and Assai - Paraná. METHODS: Latex agglutination (LA); immunochromatography (ICG); polyacrylamide gel electrophoresis (PAGE) and negative staining electron microscopy (ME) tests were used to detect the virus. For the genotyping, RT-PCR and RT-PCR-ELISA were used, respectively, for NSP4 and VP4/VP7. RESULT: Out of 124 samples there were 69 positive stool samples for RV, for at least one of the used tests, 67 of them being RV group A (RV-A). Overall, most of the RV positive stool samples came from children under thirteen years of age. However, 12 positive cases occurred in patients aged 13 years or above, including an 81-year old patient. CONCLUSION: The data showed similar electropherotypes and genotypes G, P and NSP4 of the inland wild circulating strains of RV.

Herpes simplex virus: isolation, cytopathological characterization and antiviral sensitivity

BACKGROUND: Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually self-limiting, though they can be serious, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resistant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensitivity are important in the establishment of an antivirogram for controlling the infection. OBJECTIVE: This study sought to isolate and examine the cytopathological characteristics of circulating strains of the Herpes simplex virus, from clinical specimens and their sensitivity to commercially available antiherpesvirus drugs, acyclovir, phosphonophormic acid and trifluridine. METHODS: Herpes simplex virus isolation, cytopathological features and antiviral sensitivity assays were performed in cell culture by tissue culture infectious dose or plaque forming unit assay. RESULTS: From twenty-two clinical specimens, we isolated and adapted nine strains. Overall, the cytopathic effect was detected 24 h post-infection (p.i.) and the presence of syncytia was remarkable 48 h p.i., observed after cell staining. Out of eight isolates, four developed plaques of varying sizes. All the isolates were sensitive to acyclovir, phosphonophormic and trifluridine, with the percentage of virus inhibition (%VI) ranging from 49.7-100%. CONCLUSIONS: The methodology for HSV isolation and characterization is a straightforward approach, but the drug sensitivity test, regarded as being of great practical importance, needs to be better understood. .

Detection and characterization of human rotavirus in hospitalized patients in the cities of Ponta Grossa, Londrina and Assai - PR, Brazil

Acute diarrheal disease is still one of the major public health problems worldwide. Rotaviruses (RV) are the most important viral etiologic agents and children under five years of age are the target population. OBJECTIVE: To investigate the rate of RV infection in hospitalized patients due to acute diarrhea in the cities of Ponta Grossa, Londrina and Assai - Paraná. METHODS: Latex agglutination (LA); immunochromatography (ICG); polyacrylamide gel electrophoresis (PAGE) and negative staining electron microscopy (ME) tests were used to detect the virus. For the genotyping, RT-PCR and RT-PCR-ELISA were used, respectively, for NSP4 and VP4/VP7. RESULT: Out of 124 samples there were 69 positive stool samples for RV, for at least one of the used tests, 67 of them being RV group A (RV-A). Overall, most of the RV positive stool samples came from children under thirteen years of age. However, 12 positive cases occurred in patients aged 13 years or above, including an 81-year old patient. CONCLUSION: The data showed similar electropherotypes and genotypes G, P and NSP4 of the inland wild circulating strains of RV.

The in vitro cytopathology of a porcine and the simian (SA-11) strains of rotavirus.

Rotaviruses have been implicated as the major causal agents of acute diarrhoea in mammals and fowls. Experimental rotavirus infection have been associated to a series of sub-cellular pathologic alterations leading to cell lysis which may represent key functions in the pathogenesis of the diarrhoeic disease. The current work describes the cytopathic changes in cultured MA-104 cells infected by a simian (SA-11) and a porcine (1154) rotavirus strains. Trypan blue exclusion staining showed increased cell permeability after infection by both strains, as demonstrated by cell viability. This effect was confirmed by the leakage of infected cells evaluated by chromium release. Nuclear fragmentation was observed by acridine orange and Wright staining but specific DNA cleavage was not detected. Ultrastructural changes, such as chromatin condensation, cytoplasm vacuolisation, and loss of intercellular contact were shown in infected cells for both strains. In situ terminal deoxynucleotidyl transferase (Tunel) assay did not show positive result. In conclusion, we demonstrated that both strains of rotavirus induced necrosis as the major degenerative effect.