The elicited immune responses towards the N, GN, GC and GN/GC proteins thoroughly after gene-gun immunisation were analysed and the protective abilities of the N and the GN/GC construct were tested by virus challenge. Methods Cells and viruses BHK-21 (ATCC number CCL-10) cells were maintained in Glasgow MEM (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 5% FCS, 1.3 g/l Tryptose (Difco?, Becton, Dickinson and Company, Sparks, MD), 10 mM HEPES, 1 mM sodium pyruvate, 100 U penicillin/ml and 100 ��g/ml streptomycin at 37��C/5% CO2. The working stocks of RVFV and cDNA constructs, originated from the ZH548 wild-type strain, isolated from a human case in Egypt in 1977 [24].

Viral stocks were prepared and titrated on monolayers of BHK-21 cells and the cDNA sequences are found under the GenBank accession numbers “type”:”entrez-nucleotide”,”attrs”:”text”:”AF134534″,”term_id”:”6006975″,”term_text”:”AF134534″AF134534 and “type”:”entrez-nucleotide”,”attrs”:”text”:”DQ380206″,”term_id”:”87622803″,”term_text”:”DQ380206″DQ380206[25,26]. Production of DNA vaccine For genetic immunisation and eukaryotic expression, cDNAs encoding N, GN/GC, GN and GC were inserted into pcDNA3.1/V5-His? TOPO (Invitrogen). The primer sequences used for cDNA amplification and subsequent cloning are shown in Table Table1.1. The correctness of each cDNA construct was confirmed by sequencing (MWG-Biotech) and the corresponding gene products were verified through transfection of mammalian cells followed by immunofluorescence analysis. A cDNA construct (pcDNA3.

1) encoding the N protein (PUU-N) of the Puumala hantavirus (Puumala virus Ume?/hu [GenBank: "type":"entrez-nucleotide","attrs":"text":"AY526219","term_id":"42541164","term_text":"AY526219"AY526219] [27,28] was used as a control. The preparation of gene-gun cartridges has previously been described [28]. Briefly, 50 ��g aliquots of the above plasmid DNA preparations were precipitated on 25 mg of 1 ��m gold beads and subsequently used to coat the inner wall of Tefzel tubings according to the manufacturer’s instructions (BioRad Laboratories, Hercules, CA). Each gene-gun cartridge delivered approximately 1 ��g of DNA. Table 1 Primers sequences Animal immunisation and infection Female BALB/c mice, six to eight weeks old, were used in this study. Before immunisation the mice were thoroughly shaved on the abdomen and vaccinated with cDNA encoding the antigens using a gene-gun (Helios?, BioRad Laboratories).

The cDNA was administrated four times with two to three week intervals. The primary immunisation was performed using four gene-gun cartridges Carfilzomib and the following three boosters with two cartridges. Blood samples were collected three, five, seven and nine weeks after the primary immunisation. In order to study the immune responses post infection (p.i.) and the effectiveness of the genetic vaccines, mice were injected intraperitoneally (i.p.) with RVFV diluted in sterile PBS to a final volume of 100 ��l.