Marcus Rebouças Santos

In vitro inhibition of canine distemper virus by flavonoids and phenolic acids: Implications of structural differences for antiviral design

Infection caused by canine distemper virus (CDV) is a highly contagious disease with high incidence and lethality in the canine population. Antiviral activity of flavonoids quercetin, morin, rutin and hesperidin, and phenolic cinnamic, trans-cinnamic and ferulic acids were evaluated in vitro against the CDV using the time of addition assay to determine which step of the viral replicative cycle was affected. All flavonoids displayed great viral inhibition when they were added at the times 0 (adsorption) and 1h (penetration) of the viral replicative cycle. Both quercetin and hesperidin presented antiviral activity at the time 2h (intracellular). In the other hand, cinnamic acid showed antiviral activity at the times 0 and 2h while trans-cinnamic acid showed antiviral effect at the times -1h (pre-treatment) and 0 h. Ferulic acid inhibited CDV replicative cycle at the times 0 and 1h. Our study revealed promising candidates to be considered in the treatment of CDV. Structural differences among compounds and correlation to their antiviral activity were also explored. Our analysis suggest that these compounds could be useful in order to design new antiviral drugs against CDV as well as other viruses of great meaning in veterinary medicine.

Evaluation of a synthetic peptide from the Taenia saginata 18 kDa surface/secreted oncospheral adhesion protein for serological diagnosis of bovine cysticercosis

Bovine cysticercosis is a zoonotic infection widely spread throughout Brazil, creating a burden on hygiene maintenance and the economy. Diagnosis of cysticercosis usually relies on post mortem inspection of carcasses in slaughterhouses. This detection method provides only low sensitivity. Recent advancements have improved the performance of serologic tests, such as ELISA, providing greater sensitivity and specificity. The objective of the current study was to identify and evaluate a synthetic peptide derived from the Taenia saginata 18kDa oncospheric surface protein for the diagnosis of bovine cysticercosis in ELISA. Test performance of the identified peptide was compared to an ELISA based on a heterologous crude Taenia crassiceps antigen (Tcra), widely used for the sero-diagnosis of bovine cysticercosis. Based on the primary sequence of an in silico structural model of the 18kDa protein, an epitope region designated EP1 was selected (46-WDTKDMAGYGVKKIEV-61). The peptide derived from this region yielded 91.6% (CI=80-96%) sensitivity and 90% (CI=82-95%) specificity when used in an ELISA, whereas the crude antigen yielded 70% (CI=56-8%) sensitivity and 82% (CI=73-89%) specificity. Thus, we conclude that EP1 has higher diagnostic potential for detecting bovine cysticercosis than the crude antigen Tcra.

Antibodies against Bovine herpesvirus 1 in dairy herds in the state of Espirito Santo, Brasil

Bovine herpesvirus 1 (BoHV-1) causes major losses in worldwide livestock, affecting the respiratory and reproductive tracts of bovine. In the past decades, the number of cases in Brazil has been gradually increasing. Therefore, it is important to assess the distribution of infection in different regions of the country. In the state of Espirito Santo (ES) the BoHV 1 infection rate in dairy cattle herds is unknown. Thus, the aim of this study was to detect neutralizing antibodies against BoHV-1 in serum samples from 1,161 non-vaccinated cows from 59 dairy cattle herds in 23 municipalities of the Metropolitan, North, Northwest and South macro-regions. The identification of seropositive cows was evaluated by the virus neutralization test. The results showed that of all serum samples evaluated 775 (66.75%) had neutralizing antibodies against BoHV-1. Moreover, all herds were found positive; however, the percentage of positive cows varied among regions; 49.06%, 62.15%, 67.21% and 80.04% for the Metropolitan, South, North and Northwest macro-regions, respectively. In this study, the results clearly indicate the dissemination of the viral agent in dairy cattle in the ES state, requiring the monitoring and control of diseases related to BoHV-1 infection.

Proteína bruta e energia metabolizável para codornas de corte de um a 14 dias de idade

Aiming to evaluate the effect of different levels of crude protein and metabolizable energy for quails from 1 to 14 days of age, a study was conducted using 840 quails, mixed. The birds were distributed in a factorial arrangement (2x5) consisting of 10 treatments (two levels of metabolizable energy - 2,800 and 3,000kcal/kg and 5 levels of crude protein - 22.0, 24.0, 26.0, 28.0 and 30.0%), 7 repetitions with 12 birds each. We evaluated the following parameters: feed intake, weight gain at 7 and 14 days of age, feed conversion at 14 days and viability. It was found that quail fed diets with the lowest energy level, 2,800kcal/kg, showed higher consumption. Birds fed diets with lower energy level had greater weight at 7 days of age regardless of the level of crude protein used. The weight at 14 days of age showed a quadratic response with the addition of crude protein in feed, showing that diets containing 28.86% CP provide better gain regardless of the level of energy used. The feed showed better results with the increased level of dietary CP, regardless of level of metabolizable energy used. There were no significant effects of protein and energy levels or their interaction on viability. Diets with 30.0% crude protein and 2,800 or 3,000kcal/kg metabolizable energy provide satisfactory performance results for quails from 1 to 14 days of age.

Evaluation of the genetic variability found in Brazilian commercial vaccines for infectious bronchitis virus

Infectious bronchitis virus (IBV) is currently one of the most important pathogens in the poultry industry. The H120 and Ma5 are the only viral strains approved by the Brazilian government as the constituent of vaccines. Despite the systematic vaccination in Brazil, IBV has not yet been controlled and diseases associated with this virus have been reported in vaccinated chickens. Here, we investigated the genetic variability of H120 and Ma5 strains present in the IBV vaccines from different Brazilian manufacturers. We performed DNA sequencing analyses of the S1 spike glycoprotein gene to investigate its genetic variability and the presence of viral subpopulations among vaccines, between batches, and also in each vaccine after a single passage was performed in chicken embryonated eggs. Our results revealed up to 13 amino acid substitutions among vaccines and some of them were localized in regions of the S1 glycoprotein that play a role in virus–host interaction. Secondary nucleotide peaks identified in the chromatogram for the S1 gene sequence revealed that all original vaccines (H120 and Ma5) were composed by different subpopulations of IBV. Moreover, new viral subpopulations were also found in vaccines after a single passage in chicken embryonated eggs. These findings indicate that H120 and Ma5 viral strains used in vaccines market in Brazil can still mutate very rapidly during replication, leading to amino acid substitutions in proteins involved in the stimulation of the immune response, such as the S1 glycoprotein. Therefore, our data suggest that the genetic variability of these viral strains should be taken into consideration to ensure an effective immune response against IBV.

Evaluation of the antiviral activities of Bacharis dracunculifolia and quercetin on Equid herpesvirus 1 in a murine model

Equid herpesvirus 1 (EHV-1) is a pathogen of high economic importance in equine breeding operations around the world. EHV-1 infection causes respiratory, neurologic and reproductive disease. The absence of an efficient therapy has caught the attention of the scientific community and the therapeutic activities of natural products with its antivirals effects might be effective for the diseases treatment. Herein it was evaluated the prophylactic and therapeutic potential of quercetin and ethanolic extracts of Bacharis dracunculifolia formulations compared to Penciclovir® in an in vivo EHV-1 infection model. Six to seven-week-old female C57BL/6 mice were randomly organized into fifteen groups with six animals each. Ex-1 represents the treatment post-challenge groups to assess morbidity, mortality and weight variation. Ex-2 represents the animals that received treatment for 5 days post-challenge for lesion evaluation. In Ex-3 animals were treated prior to viral challenge to assess morbidity, mortality and weight variation. All mice in the treatment groups were challenged by intranasal inoculation of 3.0 × 105 TCID50 EHV-1. The quercetin and B. dracunculifolia treatment decreased morbimortality in post-challenge treatment (Ex-1) and EHV-1 related lesions (Ex-2). Treatment prior to viral challenge (Ex-3) did not show any significant results. Based on the results of the present study, both tested formulations are promising antiviral agents for the treatment of EHV-1 infection.

Development of the multi-epitope chimeric antigen rqTSA-25 from Taenia saginata for serological diagnosis of bovine cysticercosis

Bovine cysticercosis is a worldwide distributed zoonosis caused by the larval form of Taenia saginata present in bovine muscles. The diagnosis is based on the postmortem inspection at slaughterhouses and consists of the macroscopic visualization of lesions caused by cysticercosis in muscle sites. However, parasitized animals can pass unnoticed during sanitary inspection. Thus, the objective of this study was to characterize and evaluate the performance of different peptides from different regions of T. saginata for the cysticercosis diagnosis using enzyme-linked immunosorbent assay. We generated and evaluated a new recombinant protein chimera derived from the fusion of different peptides. We selected three distinct regions of T. saginata and predicted six peptides with antigenic potential (EP2–EP7). These peptides were analyzed individually and selected for generating a new chimeric recombinant protein. The new protein was termed rqTSA-25, and its performance rates were: 93.3% sensitivity (confidence interval (CI) = 76–98%), 95.3% specificity (CI = 82–99%), 93% positive predictive value (CI = 76–98%), 95% negative predictive value (CI = 82–99%), and 95% accuracy. In the immunoblot, this protein showed no false positive or false negative reaction. Thus, the use of rqTSA-25 is recommended for the diagnosis of bovine cysticercosis.

Latent bovine herpesvirus 1 and 5 in milk from naturally infected dairy cattle

Bovine herpesvirus 1 and 5 (BoHV-1 and -5) are antigenically and genetically related and can establish latent infection. We aimed to analyze the applicability of the milk sample to detect latently BoHV-infected cattle. BoHV-1 non-vaccinated clinically healthy cows from five dairy cattle herds (herd 1, n=24; herd 2, n=39; herd 3, n=39; herd 4, n=36; herd 5, n=70) were studied. We confirmed the presence of BoHV-1, and for the first time, BoHV-5 in the milk of naturally infected dairy cattle.

Isolation and genetic stability of an infectious bronchitis virus strain (IBV)

Infectious bronchitis virus (IBV) is a member of the Coronaviridae family that represents a major cause of economic losses in the poultry industry, affecting the performance of breeders, laying hens and broiler chickens (Cavanagh, 2007). The difficulty in controlling avian infectious bronchitis (IB) is associated with the multiplicity of IBV variants that arise or are unpredictably introduced into poultry flocks. Despite the systematic use of vaccination, the disease has not yet been satisfactorily controlled (Jackwood et al., 2012).

Utilization of phage display to identify antigenic regions in the PCV2 capsid protein for the evaluation of serological responses in mice and pigs

Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51–CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.