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

Computational biology helps understand how polyploid giant cancer cells drive tumor success

Precision and organization govern the cell cycle, ensuring normal proliferation. However, some cells may undergo abnormal cell divisions (neosis) or variations of mitotic cycles (endopolyploidy). Consequently, the formation of polyploid giant cancer cells (PGCCs), critical for tumor survival, resistance, and immortalization, can occur. Newly formed cells end up accessing numerous multicellular and unicellular programs that enable metastasis, drug resistance, tumor recurrence, and self-renewal or diverse clone formation. An integrative literature review was carried out, searching articles in several sites, including: PUBMED, NCBI-PMC, and Google Academic, published in English, indexed in referenced databases and without a publication time filter, but prioritizing articles from the last 3 years, to answer the following questions: (i) “What is the current knowledge about polyploidy in tumors?”; (ii) “What are the applications of computational studies for the understanding of cancer polyploidy?”; and (iii) “How do PGCCs contribute to tumorigenesis?

Clonagem e expressão em Pichia pastoris da glicoproteína D (gD) de Herpesvírus Bovino tipo 5

Outbreaks of fatal meningoencephalitis caused by Bovine Herpesvirus type 5 (BoHV-5) cause important economic losses in national trade of bovine beef. Commercial vaccines developed against Bovine Herpesvirus type 1 can protect cattle of neurological disease caused by BoHV-5. These cross-reactions are due to its genetic and antigenic similarities of both BoHV-1 and 5. However, these vaccines can not prevent latent infection of BoHV-5, even viral spread to healthy animals. This way, new vaccines strategies are based on envelope glycoproteins and are focusing safety, economic viability and on prevention of BoHV-5 latency and spread. This glycoprotein acts in initial steps of viral infection and glycoprotein D has drawn the attention in studies carried out with BoHV-1 and other homologous herpesvirus. The gD acts on viral membrane fusion with permissive cells through glycoproteins and host receptors interactions, so it is essential for viral entry. A vaccine that is capable to stimulate specific humoral and cellular immunity against BoHV-5 must be developed. The aim of this work was the production of a recombinant truncated form of gD of BoHV-5 in yeast Pichia pastoris and its antigenicity and immunogenicity evaluation. Data show that yeast P. pastoris can express the truncated form of BoHV-5 gD to the supernatant due to its secretion of ~190mg/L of recombinant protein, simplifying protein purification. The recombinant protein was successfully recognized by antibodies of animals immunized with BoHV-5, suggesting its antigenicity and immunogenicity and that native protein characteristic are conserved. The data presented herein allow the design of future studies aiming at expression optimization and further immunogenicity evaluation, as well as its capacity to neutralize the virus in biological models and on cattle.Surtos de meningoencefalites fatais causadas pelo Herpesvírus Bovino tipo 5 (BoHV-5) ocasionam importantes perdas econômicas no comércio nacional de carne bovina. Vacinas comerciais destinadas ao Herpesvírus Bovino tipo 1 são capazes de impedir o aparecimento de sintomatologia clínica do BoHV-5. Estas reações cruzadas se devem a existência de semelhanças genéticas e antigênicas existentes entre ambos os vírus. No entanto, estas vacinas não impedem o estabelecimento da infecção latente e a disseminação do BoHV-5 para animais não imunizados. Desta forma, estratégias que visem à produção de vacinas seguras, economicamente viáveis e que possam ser capazes de impedir a latência do BoHV-5 e sua disseminação estão focadas para glicoproteínas localizadas no envelope viral e que atuam nas etapas iniciais da infecção. Dentre estas, a glicoproteína D tem se destacado em estudos realizados com BoHV-1 e outros herpesvírus homólogos. A gD atua na fusão do envelope viral com a membrana da célula permissiva no hospedeiro através de interações com receptores celulares e com outras glicoproteínas virais, sendo essencial para a entrada do capsídeo na célula. Assim, uma vacina que seja capaz de estimular respostas humorais e celulares contra esta glicoproteína deve ser desenvolvida. Os objetivos deste trabalho foram à produção da forma truncada da gD do BoHV-5 em levedura Pichia pastoris e a avaliação desta quanto a sua antigenicidade e imunogenicidade. Os resultados demonstram que a Pichia pastoris expressou a forma truncada da gD de BoHV-5 secretando para o meio de cultivo ~190mg/L da proteína recombinante, facilitando a purificação da mesma. Testada quanto a sua a sua antigenicidade e imunogenicidade, a proteína recombinante foi reconhecida por anticorpos de animais imunizados com o BoHV-5, sugerindo que características da proteína nativa foram conservadas na proteína recombinante. Os dados apresentados irão permitir o desenvolvimento de estudos 6 futuros com o objetivo de aperfeiçoar a expressão da proteína recombinante e avaliar a sua capacidade de neutralizar o vírus na espécie-alvo

Aplicação da glicoproteína D recombinante de Herpesvírus bovino 5 como antígeno em teste de imunodiagnóstico e vacina de subunidade

The new strategies in vaccine design against Bovine herpesvirus 1 and 5 are focused in the viral envelope glycoprotein, such as the gD, which acts in attachment and fusion of viral envelope with the permissive cell membranes. This glycoprotein is present at large quantities in the viral envelope and stimulates strong humoral and cellular immune responses at the host. The serological diagnostic based on immunoenzymatic assays, as the Indirect ELISA, also applies the envelope glycoproteins, once they are expose in the viral structure. Thus, the aim of this study was to apply the gD from BoHV-5 expressed in Pichia pastoris, after its antigenic characterization, as antigen in a subunit vaccine and in Indirect ELISA, aiming the control and diagnosis of BoHV-5, which is the causative agent of herpetic meningoencephalitis outbreaks, resulting in economic losses for the Brazilian livestock. The subunit vaccine developed was able to stimulate humoral immune response in mice, with development of neutralizing antibodies when the rgD was administrated alone or in combination with immunological adjuvants. The cellular immune response was observed through the increase in the mRNA expression associated with this kind of responses, as IFN-!, IL-17 and GM-CSF, when the vaccine was formulated with the oil adjuvant Emulsigen-DDA. The Indirect ELISA developed shown high sensitivity (100%) and specificity (92.9%) when compared with the virus neutralization test, which is considered the gold standard in BoHV-1 and -5 serological diagnosis. The rgD from BoHV-5 expressed in P. pastoris possess similar tridimensional conformation from the native BoHV-5 gD. The rgD is immunogenic, capable of inducing neutralizing antibodies development and the subunit vaccine developed with the rgD as antigens induces a balanced Th1/Th2 immune response. The recombinant protein antigenicity was confirmed through the Indirect ELISA, which may be used to a fast screen of BoHV-5 infected herds.Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPqAs novas estratégias no desenvolvimento de vacinas contra o Herpesvírus bovino 1 e 5 focam nas glicoproteínas do envelope viral, como a gD, a qual atua na ligação e fusão do envelope viral com a membrana de células permissivas. A gD está presente em grande quantidade no envelope e estimula resposta imune humoral e celular no hospedeiro. O diagnóstico sorológico através de teste imuno enzimático, como o ELISA Indireto, também utiliza as glicoproteínas do envelope viral, uma vez que estas estão expostas na estrutura viral. Desta forma, o objetivo deste estudo foi aplicar a gD recombinante do BoHV-5 expressa em Pichia pastoris, após caracterização antigênica, como antígeno no desenvolvimento de uma vacina de subunidade e de um ELISA Indireto visando o controle da infecção por BoHV-5, o qual é o agente responsável por surtos de meningoencefalite que ocasionam perdas econômicas na pecuária brasileira. A vacina de subunidade desenvolvida foi capaz de estimular resposta imune humoral em camundongos, com a presença de anticorpos neutralizantes, quando administrada sozinha ou com adjuvantes imunológicos. A resposta imune celular foi observada através do aumento da expressão de mRNA de citocinas associadas a este tipo de resposta, como IFN-!, IL-17 e GM-CSF, quando a vacina foi formulada com o adjuvante oleoso EmulsigenDDA. O ELISA Indireto desenvolvido demonstrou alta sensibilidade (100%) e especificidade (92.9%) quando comparado a técnica de soroneutralização viral, a qual é considerada a técnica padrão para o diagnóstico sorológico de BoHV-1 e -5. A rgD de BoHV expressa em P. pastoris possui conformação tridimensional semelhante a gD nativa de BoHV-5. A rgD é imunogênica, capaz de induzir formação de anticorpos neutralizantes e a vacina de subunidade utilizando a rgD como antígeno induz uma resposta imunológica Th1/Th2 balanceada. A antigenicidade da proteína foi confirmada com o seu uso em um ELISA Indireto, o qual pode ser utilizado para triagem rápida de rebanhos infectados com BoHV-5

Bovine herpesvirus glycoprotein D: a review of its structural characteristics and applications in vaccinology

International audienceThe viral envelope glycoprotein D from bovine herpesviruses 1 and 5 (BoHV-1 and -5), two important pathogens of cattle, is a major component of the virion and plays a critical role in the pathogenesis of herpesviruses. Glycoprotein D is essential for virus penetration into permissive cells and thus is a major target for virus neutralizing antibodies during infection. In view of its role in the induction of protective immunity, gD has been tested in new vaccine development strategies against both viruses. Subunit, DNA and vectored vaccine candidates have been developed using this glycoprotein as the primary antigen, demonstrating that gD has the capacity to induce robust virus neutralizing antibodies and strong cell-mediated immune responses, as well as protection from clinical symptoms, in target species. This review highlights the structural and functional characteristics of BoHV-1, BoHV-5 and where appropriate, Human herpesvirus gD, as well as its role in viral entry and interactions with host cell receptors. Furthermore, the interactions of gD with the host immune system are discussed. Finally, the application of this glycoprotein in new vaccine design is reviewed, taking its structural and functional characteristics into consideration

Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

Abstract Background The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50 kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis. Methods Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1 mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200 ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA). Results The expected cDNA fragment of approximately 1240 bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot. Conclusions The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity

Translational Bioinformatics Applied to the Study of Complex Diseases

Translational Bioinformatics (TBI) is defined as the union of translational medicine and bioinformatics. It emerges as a major advance in science and technology by covering everything, from the most basic database discoveries, to the development of algorithms for molecular and cellular analysis, as well as their clinical applications. This technology makes it possible to access the knowledge of scientific evidence and apply it to clinical practice. This manuscript aims to highlight the role of TBI in the study of complex diseases, as well as its application to the understanding and treatment of cancer. An integrative literature review was carried out, obtaining articles through several websites, among them: PUBMED, Science Direct, NCBI-PMC, Scientific Electronic Library Online (SciELO), and Google Academic, published in English, Spanish, and Portuguese, indexed in the referred databases and answering the following guiding question: “How does TBI provide a scientific understanding of complex diseases?” An additional effort is aimed at the dissemination, inclusion, and perpetuation of TBI knowledge from the academic environment to society, helping the study, understanding, and elucidating of complex disease mechanics and their treatment