Potential Action Mechanism and Inhibition Efficacy of Morinda citrifolia Essential Oil and Octanoic Acid against Stagonosporopsis cucurbitacearum Infestations.

The use of plant-based products has been shown to efficiently inhibit fungi-mediated diseases in agricultural crops. Here, we extracted and evaluated the composition of noni, Morinda citrifolia L., essential oil and assessed its activities against Stagonosporopsis cucurbitacearum in Cucumis melo L. Using in silico molecular approaches, potential interactions between the essential oil major components and S. cucurbitacearum tyrosine-tRNA ligase were predicted. Finally, we also measured the potential interference of plant physiology (the stomatal conductance and net photosynthesis) mediated by the application of the M. citrifolia essential oil. Chromatographic analysis revealed that octanoic acid (75.8%), hexanoic acid (12.8%), and isobutyl pent-4-enyl carbonate (3.1%) were the major essential oil compounds. Octanoic acid and noni essential oil, when used as preventive measures, reduce fungal mycelial growth at a concentration of 5 mg/mL without causing significant damage to the treated leaves, which reinforces their efficacies as preventive tools against S. cucurbitacearum. Molecular docking analyses predicted very stable interactions between the major essential oil constituents and S. cucurbitacearum tyrosine-tRNA ligase, suggesting the interference of these plant-based molecules upon enzyme activation. Octanoic acid and M. citrifolia essential oil at concentrations of 20 mg/mL decreased the stomatal conductance and net photosynthesis rate of melon plants, resulting in robust phytotoxicity. Collectively, our findings indicated that despite the phytotoxicity risks at higher concentrations, M. citrifolia essential oil and octanoic acid, have potential as alternative tools for the integrative management of S. cucurbitacearum.

Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle.

Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.

Genome Characterization of the First Mimiviruses of Lineage C Isolated in Brazil.

The family Mimiviridae, comprised by giant DNA viruses, has been increasingly studied since the isolation of the Acanthamoeba polyphaga mimivirus (APMV), in 2003. In this work, we describe the genome analysis of two new mimiviruses, each isolated from a distinct Brazilian environment. Furthermore, for the first time, we are reporting the genomic characterization of mimiviruses of group C in Brazil (Br-mimiC), where a predominance of mimiviruses from group A has been previously reported. The genomes of the Br-mimiC isolates Mimivirus gilmour (MVGM) and Mimivirus golden (MVGD) are composed of double-stranded DNA molecules of ∼1.2 Mb, each encoding more than 1,100 open reading frames. Genome functional annotations highlighted the presence of mimivirus group C hallmark genes, such as the set of seven aminoacyl-tRNA synthetases. However, the set of tRNA encoded by the Br-mimiC was distinct from those of other group C mimiviruses. Differences could also be observed in a genome synteny analysis, which demonstrated the presence of inversions and loci translocations at both extremities of Br-mimiC genomes. Both phylogenetic and phyletic analyses corroborate previous results, undoubtedly grouping the new Brazilian isolates into mimivirus group C. Finally, an updated pan-genome analysis of genus Mimivirus was performed including all new genomes available until the present moment. This last analysis showed a slight increase in the number of clusters of orthologous groups of proteins among mimiviruses of group A, with a larger increase after addition of sequences from mimiviruses of groups B and C, as well as a plateau tendency after the inclusion of the last four mimiviruses of group C, including the Br-mimiC isolates. Future prospective studies will help us to understand the genetic diversity among mimiviruses.

Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5.

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.

Soroprevalência de herpesvírus bovinos tipos 1 e/ou 5 no Estado do Rio Grande do Sul / Seroprevalence of bovine herpesvirus types 1 and/or 5 in the state of Rio Grande do Sul

Este estudo objetivou estimar a prevalência de anticorpos contra os herpesvírus bovinos tipos 1 e 5 (BoHV-1 e BoHV-5) no Estado do Rio Grande do Sul (RS), Brasil, frente a diferentes cepas de BoHV-1 e BoHV-5. As amostras de soro utilizadas foram extraídas de uma amostragem mais ampla, desenhada para estimar a prevalência de brucelose bovina no Estado. Todos os soros foram coletados de vacas com idade igual ou superior a 24 meses de idade, não vacinadas contra herpesvírus bovinos, de rebanhos de corte e leite. O cálculo amostral foi baseado em uma expectativa de prevalência média de infecção de 33 por cento, considerando-se um erro padrão não superior a 1 por cento e um intervalo de confiança de 95 por cento. Com base nesse cálculo foram examinados 2.200 soros, provenientes de 390 propriedades e 158 municípios. Os soros foram analisados na busca de anticorpos contra BoHV-1 e BoHV-5 pela técnica de soroneutralização (SN), executada frente a quatro cepas de vírus distintas: EVI123/98 e Los Angeles (BoHV-1.1); EVI88/95 (BoHV-5a) e A663 (BoHV-5b). A prevalência média de anticorpos contra o BoHV-1 e BoHV-5 nos animais amostrados foi de 29,2 por cento (642/2200); animais soropositivos foram identificados em 57,7 por cento (225/390) dos rebanhos. As estimativas de prevalência variaram de acordo com a cepa e/ou vírus utilizado para o desafio nos testes de SN. A prevalência e a sensibilidade mais altas foram obtidas quando os resultados positivos à SN frente aos quatro vírus distintos foram somados. O uso de somente um vírus de desafio na SN levaria a redução de sensibilidade de 20,4 por cento a 34,6 por cento quando comparada com os resultados positivos combinados. Estes achados evidenciam que anticorpos contra BoHV-1 e BoHV-5 estão amplamente difundidos nos rebanhos do RS, embora a prevalência em distintas regiões geográficas seja bastante variada. Os resultados obtidos nas estimativas de prevalência foram fortemente afetados pelas diferentes ...

This study was carried out to estimate the prevalence of antibodies to bovine herpesviruses types 1(BoHV-1) and 5 (BoHV-5) in the state of Rio Grande do Sul (RS), Brazil, by testing serum samples against different BoHV-1 and BoHV-5 strains. The sera examined were obtained from a larger sample designed to estimate the prevalence of bovine brucellosis within the state. All sera were collected from cows 24 months or older, not vaccinated to bovine herpesviruses, from both dairy and beef herds. The number of samples to be tested was calculated based on an estimated prevalence of infection of 33 percent, with an average standard deviation of £1 percent and a 95 percent limit of agreement. Sera from 2.200 cattle from 390 farms distributed in 158 counties were tested by serum neutralization (SN) tests in search for antibodies to the following strains: BoHV-1.1 (strains EVI123/98 and Los Angeles), BoHV-5a (strain EVI88/95) and BoHV-5b (strain A663). The overall seroprevalence to BoHV-1 and BoHV-5 in the sampled herds was 29.2 percent (642/2.200); seropositive animals were detected in 225 (57.7 percent) of the sampled farms. Prevalence estimates varied according to the virus used for challenge in SN tests. The highest prevalence and sensitivity were attained when positive SN results against the four different strains were added together. The use of only one virus for challenge in SN tests would lead to a loss in sensitivity from 20.4 percent to 34.6 percent when compared to the combined SN-positive results. These findings provide evidence that antibodies to BoHV-1 and BoHV-5 are largely spread in dairy and beef herds in RS, although prevalence in distinct geographic regions is quite variable. The results were strongly affected by the virus strains used for challenge in SN testing. This must be taken into account when performing serologic tests to detect BoHV-1 and BoHV-5 antibodies. As SN test is not capable of discriminating between antibody ...

Efficacy of a gE-deleted, bovine herpesvirus 1 (BoHV-1) inactivated vaccine / Eficácia de uma vacina inativada, gE-deletada, contra o herpesvírus bovino tipo 1 (BoHV-1)

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of economic losses in cattle. Vaccination has been widely applied to minimize losses induced by BoHV-1 infections. We have previously reported the development of a differential BoHV-1 vaccine, based on a recombinant glycoprotein E (gE)-deleted virus (265gE-). In present paper the efficacy of such recombinant was evaluated as an inactivated vaccine. Five BoHV-1 seronegative calves were vaccinated intramuscularly on day 0 and boostered 30 days later with an inactivated, oil adjuvanted vaccine containing an antigenic mass equivalent to 10(7.0) fifty per cent cell culture infectious doses (CCID50) of 265gE-. Three calves were kept as non vaccinated controls. On day 60 post vaccination both vaccinated and controls were challenged with the virulent parental strain. No clinical signs or adverse effects were seen after or during vaccination. After challenge, 2/5 vaccinated calves showed mild clinical signs of infection, whereas all non vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Serological responses were detected in all vaccinated animals after the second dose of vaccine, but not on control calves. Following corticosteroid administration in attempting to induce reactivation of the latent infection, no clinical signs were observed in vaccinated calves, whereas non vaccinated controls showed clinical signs of respiratory disease. In view of its immunogenicity and protective effect upon challenge with a virulent BoHV-1, the oil adjuvanted preparation with the inactivated 265gE- recombinant was shown to be suitable for use as a vaccine.

O Herpesvírus bovino tipo 1 (BoHV-1) é reconhecido como um importante agente de perdas econômicas em bovinos. Vacinação tem sido amplamente empregada para minimizar as perdas conseqüentes a infecções com o BoHV-1. Reportamos previamente o desenvolvimento de uma vacina diferencial para BoHV-1, baseada em um recombinante do qual a glicoproteína gE (gE) foi deletada (265gE-). No presente trabalho foi realizada a avaliação da eficácia de tal recombinante como vacina inativada. Cinco bovinos soronegativos para BoHV-1 foram vacinadas por via intramuscular no dia 0 e revacinadas 30 dias após com uma vacina inativada com adjuvante oleoso, contendo massa antigênica equivalente a 10(7.0) doses infectantes para 50 por cento dos cultivos celulares (DICC50) de 265gE-. Três animais foram mantidos como controles não vacinados. No dia 60 pós-vacinação, os animais vacinados e controles foram desafiados com a amostra virulenta parental. Nenhum sinal clínico ou efeito adverso foi observado após ou durante a vacinação. Após o desafio, 2 dos 5 animais vacinados apresentaram sinais leves de infecção, enquanto que todos os animais não vacinados apresentaram intensa rinotraqueíte e disseminaram vírus por mais tempo e em títulos mais elevados do que os animais vacinados. Respostas sorológicas foram detectadas em todos os animais vacinados depois da segunda dose de vacina, mas não nos animais do grupo controle. Após a administração de corticosteróide visando a reativação de infecções latentes, não foram observados sinais clínicos em nenhum dos 5 animais vacinados, enquanto os animais não vacinados apresentaram sinais leves de doença respiratória. Em vista de sua imunogenicidade e efeito protetor frente ao desafio com BoHV-1 virulento, a preparação oleosa com o recombinante 265gE- inativado foi demonstrada ser adequada para uso como vacina.