A rapid and simple enzymic method is described for the synthesis of [32P]phosphoenolpyruvate from... more A rapid and simple enzymic method is described for the synthesis of [32P]phosphoenolpyruvate from [32P]Pi, with a reproducible yield of 74%. The final product was shown to be a good substrate for pyruvate kinase (EC 2.7.1.40).
The antiherpes virus properties of Phyllanthus orbicularis Kunth, a Cuban-endemic medicinal plant... more The antiherpes virus properties of Phyllanthus orbicularis Kunth, a Cuban-endemic medicinal plant, have been reported previously but data on its phytochemical profile and identification of antiviral metabolites as well as their mechanisms of action are still lacking. In this work, a bioactivity-guided phytochemical analysis was performed in order to isolate anti HSV-2 compounds. P. orbicularis contained mainly phenolic acids derivatives and flavonoids. The antiviral effects were attributed to (-)-epicatechin-3-O-gallate (EC(50) = 11.7 µg/mL), procyanidins B1 and B2 (EC(50) = 32.8 µg/mL and 24.2 µg/mL, respectively) as well as oligomeric and polymeric procyanidins and their gallate derivatives. The antiviral mechanisms of the active P. orbicularis extracts and fractions were also investigated and the inhibition of several HSV-2 early replication events and DNA synthesis were observed. This is the first study of extensive fractionation and phytochemical characterization of phenolic ...
The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus ... more The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus granatensis) has resulted in numerous outbreaks with high mortality leading to the reduction, or even the disappearance, of many local populations of this wild species in the Iberian Peninsula. Currently, the available vaccines that prevent myxomatosis in domestic rabbits caused by classic strains of myxoma virus have not been assessed for use in Iberian hares. The main objective of this study was to evaluate the efficacy of commercial rabbit vaccines in Iberian hares and wild rabbits against the natural recombinant myxoma virus (ha-MYXV), bearing in mind its application in specific scenarios where capture is possible, such as genetic reserves. The study used a limited number of animals (pilot study), 15 Iberian hares and 10 wild rabbits. Hares were vaccinated with Mixohipra-FSA vaccine (Hipra) and Mixohipra-H vaccine (Hipra) using two different doses, and rabbits were vaccinated with th...
The causative viral agent of a lethal rabbit hemorrhagic disease has been purified and characteri... more The causative viral agent of a lethal rabbit hemorrhagic disease has been purified and characterized. In negative-stained preparations, the virions were icosahedral, measured 27 to 35 nm in diameter, were without an envelope, and showed 10 peripheral cup-shaped depressions. The major structural protein was 60 kilodaltons, which constitutes a unique characteristic of the Caliciviridae.
Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lys... more Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lysates and in Escherichia coli have been used to study the proteolytic processing of RHDV polyprotein encoded by ORF1. An epitope tag was used for monitoring the gene products by a specific antibody. We have identified four gene products with molecular masses of 80, 43, 73, and 60 kDa, from the amino to the carboxy terminus of the polyprotein. The amino-terminal sequences of the 43- and 73-kDa products were determined and indicated that RHDV 3C proteinase cleaved Glu-Gly peptide bonds.
Little is known about the antiviral response in molluscs. As in other invertebrates, the interfer... more Little is known about the antiviral response in molluscs. As in other invertebrates, the interferon signalling pathways have not been identified, and in fact, there is a debate on whether invertebrates possess an antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, the interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naïve mussels confer resistance in oysters to Ostreid herpesvirus 1 (OsHV-1) when oyster haemocytes are incubated with mussel haemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel haemolymph and haemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel haemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules co...
We report the complete genome sequences of two isolates (RHDV-N11 and CBVal16) of variant rabbit ... more We report the complete genome sequences of two isolates (RHDV-N11 and CBVal16) of variant rabbit hemorrhagic disease virus (RHDVb). Isolate N11 was detected in young domestic animals during a rabbit hemorrhagic disease (RHD) outbreak that occurred in 2011 on a rabbit farm in Navarra, Spain, while CBVal16 was isolated from a wild rabbit found dead in Valpaços, Northern Portugal, a year later. The viral sequences reported show 84.8-85.1 % and 78.3-78.5 % identity to RHDVAst/89 and RCV-A1 MIC-07, representative members of the pathogenic genogroup 1 RHDV and apathogenic rabbit calicivirus, respectively. In comparison with other RHDV isolates belonging to the previously known genogroups 1-6, RHDVb shows marked phenotypic differences, as it causes disease preferentially in young rabbits under 40 days of age and shows modified red blood cell agglutination profiles as well as antigenic differences that allow this variant to escape protection by the currently available vaccines.
Studies of sarcoptic mange and immunity are hampered by lack of mite sources and natural infestat... more Studies of sarcoptic mange and immunity are hampered by lack of mite sources and natural infestation models. We have investigated the clinical and pathological signs, specific IgG response and acquired immunity in naïve New Zealand White rabbits (Oryctolagus cuniculus) experimentally infested with Sarcoptes scabiei originally isolated from a clinically affected free-living European wild rabbit. Twenty rabbits were infested using two methods, direct contact for a 24 h period with a seeder rabbit simulating the natural process of infestation and application of a dressing holding approximately 1800 live mites on each hind limb (foot area) for a 24h period. Eight weeks post infestation, rabbits were treated with ivermectin and infestation cleared. Eight weeks later seventeen previously infested and four uninfested naïve controls were then re-exposed to the same S. scabiei variety using the same methods and followed for another 8 weeks. The progress of the disease was markedly more virulent in the animals infested by contact, indicating that the effective dose of mites managing to thrive and infest each rabbit by this method was higher. Nevertheless, infestation by contact resulted in partial protection to reexposure, rabbits developed high non-protective antibody titres upon reinfestation and presented severe clinical signs. However, rabbits reinfested by dressing developed lower IgG titres, and presented high levels of resistance to reinfestation, which might be due to induction of a strong local cellular response in the inoculation point that killed the mites and resulted in a lower mite effective dose, with subsequent reduced lesion development. Statistical analysis showed that sex, method of infestation and previous exposure are key factors determining the ability of rabbits to develop immunity to this disease. The rabbit-mange model developed will allow the further study of immunity and resistance to this neglected pathogen using a natural host system.
A rapid and simple enzymic method is described for the synthesis of [32P]phosphoenolpyruvate from... more A rapid and simple enzymic method is described for the synthesis of [32P]phosphoenolpyruvate from [32P]Pi, with a reproducible yield of 74%. The final product was shown to be a good substrate for pyruvate kinase (EC 2.7.1.40).
The antiherpes virus properties of Phyllanthus orbicularis Kunth, a Cuban-endemic medicinal plant... more The antiherpes virus properties of Phyllanthus orbicularis Kunth, a Cuban-endemic medicinal plant, have been reported previously but data on its phytochemical profile and identification of antiviral metabolites as well as their mechanisms of action are still lacking. In this work, a bioactivity-guided phytochemical analysis was performed in order to isolate anti HSV-2 compounds. P. orbicularis contained mainly phenolic acids derivatives and flavonoids. The antiviral effects were attributed to (-)-epicatechin-3-O-gallate (EC(50) = 11.7 µg/mL), procyanidins B1 and B2 (EC(50) = 32.8 µg/mL and 24.2 µg/mL, respectively) as well as oligomeric and polymeric procyanidins and their gallate derivatives. The antiviral mechanisms of the active P. orbicularis extracts and fractions were also investigated and the inhibition of several HSV-2 early replication events and DNA synthesis were observed. This is the first study of extensive fractionation and phytochemical characterization of phenolic ...
The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus ... more The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus granatensis) has resulted in numerous outbreaks with high mortality leading to the reduction, or even the disappearance, of many local populations of this wild species in the Iberian Peninsula. Currently, the available vaccines that prevent myxomatosis in domestic rabbits caused by classic strains of myxoma virus have not been assessed for use in Iberian hares. The main objective of this study was to evaluate the efficacy of commercial rabbit vaccines in Iberian hares and wild rabbits against the natural recombinant myxoma virus (ha-MYXV), bearing in mind its application in specific scenarios where capture is possible, such as genetic reserves. The study used a limited number of animals (pilot study), 15 Iberian hares and 10 wild rabbits. Hares were vaccinated with Mixohipra-FSA vaccine (Hipra) and Mixohipra-H vaccine (Hipra) using two different doses, and rabbits were vaccinated with th...
The causative viral agent of a lethal rabbit hemorrhagic disease has been purified and characteri... more The causative viral agent of a lethal rabbit hemorrhagic disease has been purified and characterized. In negative-stained preparations, the virions were icosahedral, measured 27 to 35 nm in diameter, were without an envelope, and showed 10 peripheral cup-shaped depressions. The major structural protein was 60 kilodaltons, which constitutes a unique characteristic of the Caliciviridae.
Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lys... more Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lysates and in Escherichia coli have been used to study the proteolytic processing of RHDV polyprotein encoded by ORF1. An epitope tag was used for monitoring the gene products by a specific antibody. We have identified four gene products with molecular masses of 80, 43, 73, and 60 kDa, from the amino to the carboxy terminus of the polyprotein. The amino-terminal sequences of the 43- and 73-kDa products were determined and indicated that RHDV 3C proteinase cleaved Glu-Gly peptide bonds.
Little is known about the antiviral response in molluscs. As in other invertebrates, the interfer... more Little is known about the antiviral response in molluscs. As in other invertebrates, the interferon signalling pathways have not been identified, and in fact, there is a debate on whether invertebrates possess an antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, the interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naïve mussels confer resistance in oysters to Ostreid herpesvirus 1 (OsHV-1) when oyster haemocytes are incubated with mussel haemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel haemolymph and haemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel haemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules co...
We report the complete genome sequences of two isolates (RHDV-N11 and CBVal16) of variant rabbit ... more We report the complete genome sequences of two isolates (RHDV-N11 and CBVal16) of variant rabbit hemorrhagic disease virus (RHDVb). Isolate N11 was detected in young domestic animals during a rabbit hemorrhagic disease (RHD) outbreak that occurred in 2011 on a rabbit farm in Navarra, Spain, while CBVal16 was isolated from a wild rabbit found dead in Valpaços, Northern Portugal, a year later. The viral sequences reported show 84.8-85.1 % and 78.3-78.5 % identity to RHDVAst/89 and RCV-A1 MIC-07, representative members of the pathogenic genogroup 1 RHDV and apathogenic rabbit calicivirus, respectively. In comparison with other RHDV isolates belonging to the previously known genogroups 1-6, RHDVb shows marked phenotypic differences, as it causes disease preferentially in young rabbits under 40 days of age and shows modified red blood cell agglutination profiles as well as antigenic differences that allow this variant to escape protection by the currently available vaccines.
Studies of sarcoptic mange and immunity are hampered by lack of mite sources and natural infestat... more Studies of sarcoptic mange and immunity are hampered by lack of mite sources and natural infestation models. We have investigated the clinical and pathological signs, specific IgG response and acquired immunity in naïve New Zealand White rabbits (Oryctolagus cuniculus) experimentally infested with Sarcoptes scabiei originally isolated from a clinically affected free-living European wild rabbit. Twenty rabbits were infested using two methods, direct contact for a 24 h period with a seeder rabbit simulating the natural process of infestation and application of a dressing holding approximately 1800 live mites on each hind limb (foot area) for a 24h period. Eight weeks post infestation, rabbits were treated with ivermectin and infestation cleared. Eight weeks later seventeen previously infested and four uninfested naïve controls were then re-exposed to the same S. scabiei variety using the same methods and followed for another 8 weeks. The progress of the disease was markedly more virulent in the animals infested by contact, indicating that the effective dose of mites managing to thrive and infest each rabbit by this method was higher. Nevertheless, infestation by contact resulted in partial protection to reexposure, rabbits developed high non-protective antibody titres upon reinfestation and presented severe clinical signs. However, rabbits reinfested by dressing developed lower IgG titres, and presented high levels of resistance to reinfestation, which might be due to induction of a strong local cellular response in the inoculation point that killed the mites and resulted in a lower mite effective dose, with subsequent reduced lesion development. Statistical analysis showed that sex, method of infestation and previous exposure are key factors determining the ability of rabbits to develop immunity to this disease. The rabbit-mange model developed will allow the further study of immunity and resistance to this neglected pathogen using a natural host system.
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Papers by Francisco Parra