Assembly of a chimeric hantavirus-like particle, containing the Araraquara nucleoprotein and the Andes glycoproteins, expressed in baculovirus system

Os hantavírus, membros da família Bunyaviridae, são os agentes infecciosos responsáveis pela Febre Hemorrágica com Síndrome Renal e pela Síndrome Cardiopulmonar por Hantavírus. São vírus com genoma constituído por três segmentos de RNA fita simples, de polaridade negativa, designados como S, M e L, que codificam, respectivamente, a nucleoproteína, as glicoproteínas G1 e G2 e a RNA polimerase dependente de RNA. Com o objetivo de estudar a montagem de pseudopartículas quiméricas de hantavírus, a proteína N do vírus Araraquara e as glicoproteínas G1 e G2 do vírus Andes foram expressas em sistema baculovírus. A microscopia confocal mostrou a colocalização das proteínas G1 e G2 com a proteína N. Pelos ensaios de imunoprecipitação e de centrifugação em gradiente de sacarose, foi observada a interação entre as proteínas N, G1 e G2. Nas análises por microscopia eletrônica de transmissão foi observada a montagem do pseudo-hantavírus quimera, com morfologia semelhante ao do vírion. O pseudo-hantavírus quimera obtido neste estudo poderá, no futuro, ser utilizado em estudos imunológicos, estruturais e morfológicos.Hantaviruses, members of the Bunyaviridae family, are the infectious agents responsible for Hemorrhagic Fever with Renal Syndrome and the Hantavirus Cardiopulmonary Syndrome. The viral genome is composed by three segments of single-stranded negative-sense RNA, designated as S, M and L, which encode, respectively, the nucleoprotein, the G1 and G2 glycoproteins, and the RNA-dependent RNA polymerase. In order to study the assembly of a chimeric hantavirus-like particle, the Araraquara nucleoprotein and the Andes glycoproteins were expressed in a baculovirus system. Confocal microscopy showed the colocalization of G1 and G2 proteins with the N protein. Immunoprecipitation assay and sucrose density gradient showed the interaction among N, G1 and G2 proteins. The transmission electron microscopy showed the hantavirus-like particle with the same morphology of the virion. The chimeric hantavirus-like particle produced in this study could be used, in the future, in immunological, structural and morphological studies

Global fitted (dotted line) SPR data of murine MX35 and Rebmab200 binding to immobilized synthetic NaPi2b epitope.

<p>MX35 (A) and Rebmab200 (B) were injected at concentrations ranging from 5 to 80 nM. After a 10 min association phase, the dissociation phase was followed for additional 10 min. Following double subtractive referencing, the curves were plotted using a 1∶1 Langmuir binding model, using Biacore T100 Evaluation Software. The solid line represents the experimental data and the dotted line the mathematical model for the binding of MX35 and Rebmab200 to the synthetic NaPi2b epitope.</p

Immunoreactivity of MX35 and Rebmab200 in tumor tissues by immunohistochemistry.

<p>Immunoreactivity of MX35 and Rebmab200 in tumor tissues by immunohistochemistry.</p

Comparison of the immuno-affinity of MX35 (left) at 1/50 dilution and Rebmab200 (right) at 1/100 dilution in parallel reactions using the same buffers and amplification and development conditions.

<p>Serial sections of serous papillary ovary adenocarcinoma (A), clear cell renal carcinoma (B), large cell poorly differentiated carcinoma of the lung (C) and invasive ductal carcinoma of the breast (D). Original magnification = 100×.</p

Comparison of ADCC activity between MX35 and Rebmab200 (stable pool) over a range of mAb concentrations.

<p>The % of cytotoxicity represents antibody-mediated cell lysis measured by release of ⁵¹Cr from labeled ovarian cancer cells (OVCAR-3). Effector cells were obtained from donated human peripheral blood. Rebmab100 was used as a positive control, and Zenapax (Roche) was used as a negative control. The assay was repeated with MCF-7 cells, a NaPi2b negative and Le^Y positive (Rebmab100 antigen) tumor cell line, showing no ADCC results (data not shown).</p

Immunoreactivity of MX35 and Rebmab200 in live cells by flow cytometry.

<p>Immunoreactivity of MX35 and Rebmab200 in live cells by flow cytometry.</p

Immunoglobulin HC and LC mRNA levels and productivity of the most representative Rebmab200 clones as determined by RT-qPCR.

<p><b>GAPDH was used as a reference gene.</b> Vector represents PER.C6®cells transfected with mock vector; PER.C6® corresponds to the parental cell. Error bars represent the standard deviation of triplicates. Qp represents specific productivity levels.</p

Infection kinetics of human adenovirus serotype 41 in HEK 293 cells

The purpose of this work was to acquire an overview of the infectious cycle of HAdV-41 in permissive HEK 293 cells and compare it to that observed with the prototype of the genus, Human adenovirus C HAdV-2. HEK 293 cells were infected with each virus separately and were harvested every 12 h for seven days. Infection kinetics were analysed using confocal and electronic microscopy. The results show that, when properly cultivated, HAdV-41 was not fastidious. It had a longer multiplication cycle, which resulted in the release of complete viral particles and viral stocks reached high titres. After 60 h of infection, the export of viral proteins from the infected cell to the extracellular milieu was observed, with a pattern similar to that previously described for HAdV-2 penton-base trafficking after 30 h of infection. HAdV-41 had a non-lytic cycle and the infection spread from the first infected cell to its neighbours. The release process of the viral particles is unknown. The results observed for HAdV-41 infection in HEK 293 cells show how different this virus is from the prototype HAdV-2 and provides information for the development of this vector for use in gene therapy