Notification of the first isolation of Cacipacore virus in a human in the State of Rondônia , Brazil

Flavivirus is a genus of arthropod-transmitted viruses of the family Flaviviridae, and in Brazil, up to eleven different Flavivirus have been isolated. We collected blood from farmers in the municipality of Theobroma, which is located 320km from the City of Porto Velho, the former capital of the Brazilian state of Rondônia. For viral isolation, we used newborn mouse brain, followed by RT-PCR with specific universal Flavivirus primers. We obtained fragments 958bp and 800bp in length. Based on BLAsT, these sequences were 91% similar to a sequence of Cacipacore virus.

The genus Flavivirus is composed of arthropod-transmitted viruses belonging to the Flaviviridae family 1 .Flavivirus comprises more than 70 viruses sharing common antigenic determinants and contains the first human virus to be isolated, yellow fever virus, which is also the prototype virus of the genus.Eleven flaviviruses are known to occur in Brazil: Bussuquara virus (BsGV), Cacipacore virus (CPCV), dengue virus (DENV-1, 2, 3, and 4), Iguape virus (IGUV), Ilheus virus (ILHV), Rocio virus (ROCV), saint Louis encephalitis virus (sLEV) and yellow fever virus (YFV).Most of the Brazilian flaviviruses are maintained in nature as sylvatic zoonoses that occasionally infect humans and domestic animals in rural and periurban areas 2 .The differential clinical diagnosis among arboviruses can be difficult, principally in the acute phase of the infection where disease symptoms are similar.Traditionally, the diagnosis of Flavivirus infection has been done by virus isolation or serological testing.To overcome such problems, reverse transcription-polymerase chain reaction (RT-PCR) methods using universal primers have been described for certain flaviviruses [3][4][5][6][7] .
The patient under study was a farmworker thirty three years of age, originating from the municipality of Theobroma, which is located 320km from the City of Porto Velho, the former capital of the Brazilian state of Rondônia.He was admitted to the Intensive Treatment Unit of the Dr. Ary Pinheiro Hospital with suspected diagnoses of both leptospirosis and yellow fever.
Blood samples were collected in the intensive treatment unit and sent to Institute for Research in Tropical Diseases to be used in viral isolation using newborn mouse brain.After isolation RT-PCR with specific universal Flavivirus primers would be conducted for the identification of the viruses via the electrophoretic migration pattern of the amplicons, nucleotide sequencing and comparison with published sequences.

Viral isolation
Five microliters of the patient's serum was inoculated into the brains of six newborn mice.The mice were observed for approximately seven days for signs of encephalitis.Diseased mice were stored at -80 o C. The brain was eventually macerated and diluted into Leibovitz's-15 media in a 1/20 dilution.Next, 300μL of this mixture was inoculated in a monolayer of C6/36 cultures of Aedes albopictus cells 8 .Uninfected cell culture supernatant was used as a negative control.strains of yellow fever virus and dengue virus type 3 were used as positive controls to test the specificity of the assays and were grown and maintained with Leibovitz's-15 medium (Leibovitz) containing 10% fetal bovine serum and 100mg/ml of antibiotic-antimycotic.The infected cultures were observed for seven days, and 500μl of supernatant was collected for viral RNA extraction.Cultures were maintained in a humidity controlled atmosphere at 28 o C until the supernatants were collected.

Viral ribonucleic acid (RNA) extraction
RNA was extracted from the supernatants of infected C6/36 cultures using TRIzol (Life Technologies, UsA) with modifications to the original protocol 9 .

Reverse transcription -polymerase chain reaction
As previously described by De Moraes Bronzoni et al. 10 , PCR was performed using genus-specific rapid detection of the Flavivirus genus, which amplifies the Ns5 gene, and using the mosquitoborne Flavivirus universal primer pair, selected by Tanaka et al. 7 , for maximum homology with six species of non-American flaviviruses based on the original sequence of the yellow fever virus (17D vaccine).
Purification, cloning and nucleotide sequencing Amplicons were purified from a 2% agarose gel using Perfectprep® Gel cleanup Kit (Eppendorf, UsA).The DNA fragments were cloned using the pGEM T Easy cloning kit (Promega-UsA).The cloned DNA fragment was sequenced using the DYEnamic ET DYE  terminator cycle sequencing kit in a mega BACE 1000 sequencer (Amersham Pharmacia Biotech, UsA).
Amplicons of 958bp and 800bp were obtained by RT-PCR using the genus-specific, Flavivirus primer pairs FG1/FG2 and (Figure 1) FLAV1/FLAV2, respectively (Figure 2).Results revealed that the virus isolated from the patient showed 91% sequence similarity to other isolates of Cacipacore BeAn 4073 when using BLAsT and the sequences available in GenBank (Figure 3).

FIGURE 3 -
FIGURE 3 -Alignment of nucleotide sequence of Cacipacore virus (CACV) isolated in Theobroma City, in the State of Rondônia, with strain Cacipacore BeAn 4073 virus obtained by BLAST.