Yellow fever (YF) is an haemorragic disease caused by a virus and transmitted by mosquitoes through two distinct cycles: the urban YF, transmitted by Aedes aegypti and the sylvatic YF, maintained in a enzootic cycle by Haemagogus and Sabethes mosquitoes with monkeys as main hosts . No cases of urban YF have been reported in Brazil since 1942 . The sylvatic YF is mostly restricted to wild and rural areas but recent outbreaks amongst human visitors and travelers together with the reinfestation of urban areas with the vector mosquito Aedes aegypti have concerned health authorities about the reurbanization of YF.
The main mechanisms for YF control consists of vaccination and insect vector control in urban areas. Yet, in 2008, 228 YF epizootic cases were reported and 64 cases of dead monkeys ocurred just in January. These are more than the 104 YF epizootic cases and the 17 cases of dead monkeys during the whole year of 2007. Until July of the same year, 45 cases of YF amongst humans were confirmed with 25 deaths, which represents 55,6% case fatality rate . Similar to what happens to other flavivirus diseases, the investment on improved diagnosis techniques for YF with faster and precise methods is crucial for the early detection and correct identification of yellow fever infection for prevention and control of disease spread by public health authorities besides correct epidemiological reports .
Although clinical diagnosis is sufficient during an epidemic, laboratory diagnosis is the definite method to confirm yellow fever infection mainly in sporadic cases because yellow fever main symptoms may be confused with a broad range of related diseases that vary from severe malaria to dengue or leptospirosis. Viral isolation in mosquito cells cultures is a sensitive technique for the first days of infection, during the viremic period of the disease, but currently yellow fever diagnosis is based on serology, mainly enzyme-linked immunosorbent assays (ELISA) . Yet, serological tests still use the whole virus as the antigen pool which could be improved with specificity by the use of antigenic parts of the virus, minimizing the risk of cross reactions with other flavivirus and risk of infection by health professionals.
Yellow fever virus (YFV) is an enveloped virus with a positive sense, single stranded RNA genome of 10,862 bases coding for a single ORF of 10,233 bp. This ORF encodes three structural proteins (Capsid, pM, and E) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) . Among the viral proteins, the E protein is the most studied one, due to its high antigenic potencial.
E protein is involved in many events, such as receptor binding site for viral attachment , fusion, penetration, hemagglutination, host range and cell tropism . It also has an important role in immunological anti-virus response, eliciting neutralizing antibodies and inducing protective response .
Native E protein presents itself as homodimers and its activity is intimately linked to its structure that suffers conformation rearrangements changing the native homodimer into a fusogenic homotrimer after entering cells by receptor-mediated endocytosis . The conformational change occurs in the lower pH environment of the endosome where viral lipid envelope fusion with endossomal membrane, releasing the nucleocapsids into the cells cytoplasm . Each E protein monomer has a molecular mass of 50-55 kDa and presents 3 distinct domains: domain I, II and III. Domain III is the immunoglobulin-like receptor binding domain  and is recognized by virus-neutralizing antibodies, being considered a target for diagnosis assays .
On the purpose of isolating viral parts and expressing them separately, different heterologous expression systems may be used and within these, the Baculovirus Expression System is one of the most popular and efficient.
Baculoviruses are large (30-60 × 250-300 nm), rod-shaped, double-stranded DNA (80-180 Kbp) viruses that are highly specific and only capable of replication in arthropod hosts . This is why baculoviruses were first studied as biological control agents to protect crops and forests . The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is the most studied baculovirus at the molecular level and most expression vectors are based on this virus . There are many advantages of using the Baculovirus Expression System such as high expression levels and post-translational modifications that allows the expressed heterologous proteins to be correctly folded and biologically active .
In order to evaluate the potencial antigenic activity of the Yellow fever E protein, in this study, we constructed a recombinant baculovirus containing a cDNA encoding the env gene of yellow fever virus. This recombinant virus was used to infect insect cells and larvae of susceptible insects and the protein produced was tested for its antigenicity.