PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

Bovine viral diarrhea virus (BVDV) can cause acute and persistent infections in cattle, resulting in significant economic losses to the livestock industry each year. Targeted antiviral therapy is an effective strategy. This study was based on network pharmacology, molecular docking techniques and in vitro studies to investigate quercetin’s mechanism in treating bovine viral diarrhea/mucosal disease (BVD-MD). The network topology analysis was carried out using Cytoscape 3.9.0 software to construct the network of “Chinese medicine ingredients-target-diseases”. Protein interactions were explored and analyzed using the String system (PPI). GO and KEGG pathway analysis of the intersected targets was performed using Bioconductor software. The molecular docking and molecular dynamics simulation methods were used to reveal the degree of binding of quercetin to key target genes. Western blot and indirect immunofluorescence were used to characterize the antiviral effects of quercetin. This study utilized network pharmacological analysis, identifying 22 targets associated with BVD-MD. The results of the KEGG pathway showed that quercetin was closely related to Ras and MAPK signaling pathways of BVD-MD. Molecular docking results showed that SRC, NS5B, NOX4 and XDH were the key targets of quercetin in the treatment of BVD-MD. Through network pharmacology, molecular docking and in vitro experiments, quercetin was demonstrated to combat bovine viral diarrhea mucosal disease through key targets of SRC, MAPK1, GSK3B, NS5B and E2. Molecular dynamics analysis showed that quercetin exhibited complex stability with NS5B. This study provides a theoretical and experimental basis for quercetin treatment of BVD-MD and later drug development.

• BVD-MD quercetin web-based drug screening molecular docking antiviral

Abstract in Portuguese:

Bovine viral diarrhea virus (BVDV) can cause acute and persistent infections in cattle, resulting in significant economic losses to the livestock industry each year. Targeted antiviral therapy is an effective strategy. This study was based on network pharmacology, molecular docking techniques and in vitro studies to investigate quercetin’s mechanism in treating bovine viral diarrhea/mucosal disease (BVD-MD). The network topology analysis was carried out using Cytoscape 3.9.0 software to construct the network of “Chinese medicine ingredients-target-diseases”. Protein interactions were explored and analyzed using the String system (PPI). GO and KEGG pathway analysis of the intersected targets was performed using Bioconductor software. The molecular docking and molecular dynamics simulation methods were used to reveal the degree of binding of quercetin to key target genes. Western blot and indirect immunofluorescence were used to characterize the antiviral effects of quercetin. This study utilized network pharmacological analysis, identifying 22 targets associated with BVD-MD. The results of the KEGG pathway showed that quercetin was closely related to Ras and MAPK signaling pathways of BVD-MD. Molecular docking results showed that SRC, NS5B, NOX4 and XDH were the key targets of quercetin in the treatment of BVD-MD. Through network pharmacology, molecular docking and in vitro experiments, quercetin was demonstrated to combat bovine viral diarrhea mucosal disease through key targets of SRC, MAPK1, GSK3B, NS5B and E2. Molecular dynamics analysis showed that quercetin exhibited complex stability with NS5B. This study provides a theoretical and experimental basis for quercetin treatment of BVD-MD and later drug development.

• BVD-MD quercetin web-based drug screening molecular docking antiviral

Abstract in English:

The search for an alternative to synthetic drugs have revealed discoveries in the field of pharmacology and, according to melittin and apamin, two components of bee venom which have been described were with various pharmacological actions.This study aimed to evaluate the in vitro antiviral and virucidal capabilities of these components. Therefore, after verification of their toxic doses by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, MDBK cells (Madin Darby Bovine Kidney) have been cultivated in microplates and treated with different concentrations of apamin, melittin and its association. This treatment occurred before and after infection with MOI (multiplicity of infection) 0.1 of cytopathogenic strains of bovine herpesvirus type 1 (BoHV-1) strain Los Angeles and bovine viral diarrhea virus (BVDV) strain NADL. After incubation for 72 hours, 37°C, the cells were submitted to MTT assay to estimate cell viability. In parallel experiments, plates were subjected to the same procedure suffered freezing and thawing cycle the cells to rupture the same and measurement of viral titers. The virucidal assay was performed by incubating suspension of bovine herpesvirus type-1 and BVDV with apamin solutions, melittin and association for 24 hours at 37°C and 22°C. The viral titer was evaluated at 0 hours, 1, 2, 4, 8 and 24 hours of incubation. The cytotoxic concentration to 50% of the cells (CC50) of melittin was 2.32g/mL and apamin did not show toxicity at the greater concentration tested (100μg/mL). There was antiviral effect of melittin on bovine herpesvirus type-1, especially at a concentration of 2μg/mL, where was observed 85.96% cell viability when treatment was performed before the infection and 86.78% viability when the treatment was carried out after infection. There was also a 90% reduction of viral particles of bovine herpesvirus type-1. In lower concentrations (1 and 1.5μg/mL) melittin no antiviral activity because cell viability was low, showing cytopathic effect of the virus. At the association two substances there were a decrease in the title of BVDV and there was higher cell viability when compared to the isolated action of the compounds of this virus. This is confirmed in the virucidal activity, since there was a decrease of 90% of the viral particles of BVDV with the combination of the two compounds of bee venom. Acting individually, melittin showed antiviral effect and virucidal against for BoHV-1, zeroing its title in just 2 hours at 37°C. It is concluded that melittin has antiviral and virucidal action against the BoHV-1 and its association with apamin potentiate its effects against BVDV.

• Melittin apamin bovine herpesvirus type 1 bovine viral diarrhea virus antimicrobial cattle bee venom BoHV-1 BVDV viroses

Abstract in Portuguese:

A busca por alternativa aos fármacos sintéticos têm revelado descobertas no campo da farmacologia e, nesse sentido, melitina e apamina, dois constituintes do veneno de abelhas, foram descritas com várias ações farmacológicas. Este estudo objetivou avaliar in vitro as capacidades antiviral e virucida destes componentes. Para tanto, células MDBK (Madin Darby Bovine Kidney), após verificação das respectivas doses tóxicas por ensaio MTT ((3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo), foram cultivadas em microplacas e tratadas com diferentes concentrações de apamina, melitina e sua associação. Esse tratamento ocorreu antes e após a infecção com 0,1 MOI (multiplicidade de infecção) de cepas citopatogênicas de herpesvírus bovino tipo 1 (BoHV-1) cepa Los Angeles e vírus da diarreia viral bovina (BVDV) cepa NADL. Após incubação por 72 horas, 37oC, as células foram submetidas ao ensaio MTT para estimativa da viabilidade celular. Em experimento paralelo, placas que foram submetidas ao mesmo procedimento sofreram ciclo de congelamento e descongelamento das células, para rompimento das mesmas e mensuração dos títulos virais. O ensaio virucida foi realizado incubando-se suspensões de BoHV-1 e BVDV com as soluções de apamina, melitina e associação por 24 horas a 37oC e 22oC. O título viral foi avaliado às 0 horas, 1, 2, 4, 8 e 24 horas de incubação. A concentração citotóxica para 50% das células (CC50) de melitina foi 2,32 μg/ml e apamina não demonstrou toxicidade à maior concentração testada (100μg/ml). Houve efeito antiviral da melitina sobre BoHV-1, especialmente na concentração de 2μg/ml, onde observou-se 85,96% de viabilidade celular quando o tratamento foi realizado antes da infecção e 86,78% de viabilidade quando o tratamento foi realizado após a infecção. Houve ainda redução de 90% das partículas virais de BoHV-1. Em menores concentrações (1 e 1,5μg/ml) de melitina não houve atividade antiviral, pois a viabilidade celular foi baixa, demonstrando efeito citopático do vírus. Na associação das duas substâncias houve queda no título de BVDV e observou-se maior viabilidade celular quando comparados à ação isolada dos composto sobre este vírus. Isso se confirma na atividade virucida, uma vez que houve decréscimo de 90% das partículas virais de BVDV com a associação dos dois compostos do veneno de abelhas. Atuando individualmente, melitina apresentou efeito antiviral e virucida frente ao BoHV-1, zerando seu título em apenas 2 horas a 37oC. Conclui-se que melitina tem ação antiviral e virucida frente ao BoHV-1 e sua associação com apamina potencializou seus efeitos frente ao BVDV.

• Melitina apamina herpesvírus bovino tipo 1 vírus da diarreia viral bovina bovinos antimicrobiano veneno de abelha BoHV-1 BVDV viroses

Abstract in English:

ABSTRACT.- Peter C.M., Picoli T., Zani J.L., Latosinski G.S., Lima M., Vargas G.D., Hübner S.O. & Fischer G. 2017. [Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and Jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV).] Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas jataí (Tetragonisca angustula) frente ao Herpersvírus Bovino tipo 1 (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Pesquisa Veterinária Brasileira 37(7):667-675. Departamento de Veterinária Preventiva, Universidade Federal de Pelotas, Campus Capão do Leão, Avenida Eliseu Maciel s/n, Jardim América, Capão do Leão, RS 96900-010, Brazil. E-mail: cristina_peter@hotmail.com Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.

• Tetragonisca angustula herpesvirus type-1 Viral Diarrhea Virus BVDV BoHV-1 BVDV Herpersvírus Bovino tipo 1 Vírus da Diarreia Viral Bovina

Abstract in Portuguese:

RESUMO.- Peter C.M., Picoli T., Zani J.L., Latosinski G.S., Lima M., Vargas G.D., Hübner S.O. & Fischer G. 2017. [Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and Jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV).] Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas jataí (Tetragonisca angustula) frente ao Herpersvírus Bovino tipo 1 (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Pesquisa Veterinária Brasileira 37(7):667-675. Departamento de Veterinária Preventiva, Universidade Federal de Pelotas, Campus Capão do Leão, Avenida Eliseu Maciel s/n, Jardim América, Capão do Leão, RS 96900-010, Brazil. E-mail: cristina_peter@hotmail.com Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57µg/mL), quando comparado aos extratos verde (0,78µg/mL) e marrom (0,39µg/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.

Abstract in English:

ABSTRACT.- Dezengrini R., Silva S.C., Weiss M., Kreutz L.C., Weiblen R. & Flores E.F. 2010. [Activity of three antiviral drugs against bovine herpesviruses 1, 2 and 5 in cell culture.] Atividade de três drogas antivirais sobre os herpesvírus bovino tipos 1, 2 e 5 em cultivo celular. Pesquisa Veterinária Brasileira 30(10):855-860. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Av. Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com The activity of three anti-herpetic drugs (Acyclovir [ACV], Gancyclovir [GCV] and Foscarnet [PFA]) was tested against bovine herpesvirus 1 (BoHV-1), 2 (BoHV-2) and 5 (BoHV-5) in vitro using the plaque reduction assay. Different drug concentrations were tested against one hundred 50% tissue culture infectious dose (TCID50) of the respective viruses. Drug concentrations lower than 200µg/mL resulted in viability rates of more than 80% for MDBK and Hep2 cells in the MTT test (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The selectivity index (IS) of the drugs was calculated dividing the concentration of the drug that is cytotoxic for 50% of the cells (CC50) by the concentration of the drug that was effective in reducing by 50% the number of viral plaques (EC50) for the three herpesviruses. Thus, ACV was shown to be moderately active against BoHV-1 (EC50: 112.9mg/mL; IS: 4.5), BoHV-2 (EC50: 114.2mg/mL; IS: 4.5) and BoHV-5 (EC50: 96.9mg/mL; IS: 5.3). GCV was effective against BoHV-2 (EC50: 33.5mg/mL; IS: 16.6), moderately effective against BoHV-5 (EC50: 123.2mg/mL; IS: 4.5) and poorly active against BoHV-1 (EC50: 335.8mg/mL; IS: 1.7). PFA exhibited the highest antiviral activity, being the only drug that, at concentration of 100mg/mL, completely inhibited plaque formation by all three viruses. PFA was the most effective in vitro against BoHV-1 (EC50: 29.5mg/mL; IS: 42.2), BoHV-2 (EC50: 45.2mg/mL; IS: 27.6) and BoHV-5 (EC50: 7.8mg/mL; IS: 160.6). Thus, the results indicate that PFA is a promising candidate for experimental therapeutic testing in vivo against bovine herpesviruses.

• Antivirals BoHV-1 BoHV-2 BoHV-5 Foscarnet Gancyclovir Acyclovir antivirais Ganciclovir Aciclovir

Abstract in Portuguese:

RESUMO.- Dezengrini R., Silva S.C., Weiss M., Kreutz L.C., Weiblen R. & Flores E.F. 2010. [Activity of three antiviral drugs against bovine herpesviruses 1, 2 and 5 in cell culture.] Atividade de três drogas antivirais sobre os herpesvírus bovino tipos 1, 2 e 5 em cultivo celular. Pesquisa Veterinária Brasileira 30(10):855-860. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Av. Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com A atividade de três fármacos antivirais (Aciclovir [ACV], Ganciclovir [GCV] e Foscarnet [PFA]) foi testada in vitro frente aos herpesvírus bovino tipos 1 (BoHV-1), 2 (BoHV-2) e 5 (BoHV-5). Para isso, utilizou-se o teste de redução de placas virais em cultivo celular, testando-se diferentes concentrações dos fármacos frente a 100 doses infectantes para 50% dos cultivos celulares (DICC50) dos respectivos vírus. Pelo teste de MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), verificou-se que concentrações inferiores a 200µg/mL dos três antivirais resultaram em índices de viabilidade de células MDBK e Hep2 superiores a 80%. Com base na concentração citotóxica para 50% das células (CC50) e na concentração dos fármacos efetiva para inibir em 50% o número de placas virais (EC50), calculou-se o índice de seletividade (IS) dos antivirais para os três herpesvírus. Assim, o ACV demonstrou ser moderadamente ativo frente ao BoHV-1 (EC50: 112,9mg/mL e IS: 4,5), ao BoHV-2 (EC50: 114,2 mg/mL e IS: 4,5) e BoHV-5 (EC50: 96,9mg/mL e IS: 5,3). O GCV apresentou atividade moderada frente ao BoHV-2 (EC50: 33,5mg/mL e IS: 16,6) e, em menor grau, contra o BoHV-5 (EC50: 123,2mg/mL e IS: 4,5), sendo ineficaz frente ao BoHV-1 (EC50: 335,8mg/mL e IS: 1,7). O PFA apresentou atividade antiviral mais pronunciada, sendo o único fármaco que, na concentração de 100µg/mL, inibiu completamente a produção de placas pelos três vírus testados. O PFA foi o mais efetivo in vitro frente ao BoHV-1 (EC50: 29,5mg/mL e IS: 42,2), ao BoHV-2 (EC50: 45,2mg/mL e IS: 27,6) e ao BoHV-5 (EC50: 7,8mg/mL e IS: 160,6). Portanto, os resultados obtidos indicam que o PFA pode se constituir em um candidato para terapia experimental de infecções pelos herpesvírus de bovinos in vivo.

Abstract in English:

ABSTRACT.- Dezengrini R., Silva S.C., Weiss M., Oliveira M.S., Traesel C.K., Weiblen R. & Flores E.F. 2010. [Effects of Foscarnet on the infection by bovine herpesviruses 1 and 5 in rabbits.] Efeitos do Foscarnet sobre a infecção pelos herpesvírus bovino tipos 1 e 5 em coelhos. Pesquisa Veterinária Brasileira 30(8):623-630. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Av, Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com The activity of Foscarnet (PFA) against three bovine herpesviruses (BoHVs) was previously demonstrated in cell culture. Herein we evaluated the effects of PFA on the infection and disease by BoHV-1 and BoHV-5 in a rabbit model. Rabbits inoculated with BoHV-5 in the conjunctival sac (IC) and treated with PFA (100 mg/kg/day) from day 1 to 17 post-inoculation (pi) shed less virus between days 2 and 6 pi comparing to untreated controls; this difference was significant at day 3 pi [F(9,108) = 2,23; P<0.03]. The morbidity and mortality rates of rabbits inoculated with BoHV-5 IC or intranasally (IN) were also significantly reduced in PFA-treated rabbits (50%; 11/22) comparing to untreated controls (95.4%; 21/22) (P<0.0008). In rabbits inoculated IC with BoHV-1, a reduction in virus shedding was observed in PFA-treated animals between days 1 and 4 pi; 6 and 7 pi. In addition, PFA-treated rabbits presented a longer incubation period and a shorter clinical course comparing to untreated controls (P<0.005 and P<0.04, respectively). The frequency and severity of ocular signs were also reduced in the PFA-treated group. These results demonstrate that PFA is effective against BoHV-1 and BoHV-5 in vivo and open the way towards its use in experimental therapy of herpetic infections in domestic animals.

• Antivirals BoHV-1 BoHV-5 PFA bovine herpesviruses 1 bovine herpesviruses 5 Foscarnet herpesvírus bovino tipo 1 herpesvírus bovino tipo 5

Abstract in Portuguese:

RESUMO.- Dezengrini R., Silva S.C., Weiss M., Oliveira M.S., Traesel C.K., Weiblen R. & Flores E.F. 2010. [Effects of Foscarnet on the infection by bovine herpesviruses 1 and 5 in rabbits.] Efeitos do Foscarnet sobre a infecção pelos herpesvírus bovino tipos 1 e 5 em coelhos. Pesquisa Veterinária Brasileira 30(8):623-630. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Av, Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com