BR102013011247A2 - PROCEDURE FOR CONSTRUCTION OF INFECTIOUS RNAS FROM DENGUE TYPE 3 VIRUS CLONES CDNAS - Google Patents

Abstract

PROCESS FOR THE CONSTRUCTION OF INFECTIOUS RNES FROM DENGUE TYPE 3 VIRUS CLONES CDNAS, PROCESS FOR THE CONSTRUCTION OF INFECTIOUS RNES FROM DENGUE TYPE 3 VIRUS AND CHEMICAL CLONES CDRES AND ROCUS RERUS AND REFERRAL. This invention describes a process for constructing infectious RNAs from cDNAs of clones of dengue virus 3 (DENV-3) and chimeric clones of DENV-3 and the Rocio virus (ROCV), as well as the obtained RNAs. These viral constructs are a tool for studying the pathogenesis of DENV-3 and ROCV, as well as providing subsidies for future vaccines.

Description

PROCEDURE FOR CONSTRUCTION OF INFECTIOUS RNAS FROM DENGUE TYPE 3 VIRUS CLONES CDNAS

Field of the Invention: This invention describes a process for constructing infectious RNAs from cDNAs of dengue virus type 3 (DENV-3) clones and chimeric DENV-3 and Rocio virus (ROCV) clones, as well as RNAs. obtained.

These viral constructs are a tool for studying the pathogenesis of DENV-3 and ROCV, as well as providing input for future vaccines.

BACKGROUND OF THE INVENTION: The first disease-causing virus in humans was discovered over a century ago when Walter Reed (1901) demonstrated that serum filterable agents from yellow fever individuals caused this disease in healthy individuals and that these agents were transmitted. through an arthropod vector (hematophagous mosquitoes).

Subsequently, the etiological agent of this viral disease was named yellow fever virus (YFV). YFV has become the prototype of the Flaviviridae family, which still comprises the genera Pestivirus, Hepacivirus and Flavivirus.

Flaviviruses have spherical viral particles, 50-60 nm in diameter, consisting of a nucleocapsid containing an RNA, which is surrounded by a bilipid membrane containing anchored surface viral glycoproteins. The genome consists of a single strand of positive polarity RNA of approximately 11 kilobases, which has cap structure (m7G5'ppp5 'A) at the 5' end, but contains no poly A tail at the 3 'end. Viral RNA has a single open reading frame (ORF) that encodes a large polyprotein, which is further cleaved by cellular and viral proteases into 3 structural proteins (C-preM-E) and 7 nonstructural proteins (NS1). , NS2A, NS2B, NS3, NS4A, NS4B and NS5).

At the 5 'and 3' ends flanking the ORF there are non-coding regions (RNC5 ', RNC3') having about 100 and 400 nucleotides, respectively. These regions have conserved sequences and secondary RNA structures that drive viral replication, translation and packaging processes. The virus comes into contact with cells through the interaction of protein E with cell surface receptors. Flaviviruses may utilize multiple receptors, depending on different host species for cellular infection. Infection of intradermal dendritic cells is particularly important, making it one of the first target cells in infection. After the virus binds with its cellular receptor, the virus enters the cell by endocytosis.

Endosomes containing viral particles fuse with lysosomes, leading to a decrease in intra-endosomal pH and enhancing conformational changes in protein E. Thus, the viral envelope fuses with the endosomal vesicle membrane and, consequently, disassembly. of viral particle and genome release in cytoplasm. After the release of viral RNA into the cytoplasm, translation and processing of the polyprotein begins. Virion assembly occurs in association with endoplasmic reticulum (ER) membranes. Protein C, together with RNA, forms the nucleocapsid that binds to the cytoplasmic portion of ER membrane-anchored viral glycoproteins, promoting the assembly of viral particles by budding into the lumen. Subsequently, viral particles are released from the cell via the exocytic pathway, in which the immature viral particle undergoes post-translational modifications. Dengue is a non-contagious infectious disease caused by dengue virus (DENV), which is a flavivirus transmitted to humans by the bite of blood-eating mosquitoes, especially Aedes aegypti. Other Aedes mosquitoes, such as Aedes albopictus and Aedes africanus, have been listed as secondary transmitters in Asia and Africa, respectively.

Dengue is currently considered the most widespread arbovirus (arthropod-borne viral disease) in the world, occurring on all continents. Dengue presents in three clinical forms, namely: an undifferentiated febrile disease, a classic dengue fever (DH) and a hemorrhagic dengue fever with or without shock (DHF / DSS). DF presents as a self-limiting acute febrile illness that lasts for approximately 4 to 5 days. The disease begins abruptly, with high fever, retro-orbital pain, varying degree headache, muscle and joint pain, and maculopapular eruption. Also, thrombocytopenia and moderate-grade hemorrhagic phenomena are observed.

In DHF / DSS, in addition to the initial symptoms of DF, thrombocytopenia and hemorrhagic phenomena intensify. Between the second and fourth day of the disease, interstitial fluid extravasation syndrome, characteristic of DHF / DSS, can be observed, leading to a decrease in intravascular plasma volume and consequent hypovolemic shock that may progress to death.

Classic serological methods, such as hemagglutination inhibition (HI), complement fixation (CF) or neutralization (NT), can be used to diagnose dengue infection.

These tests are performed with paired samples, one collected at the onset of symptoms and the other at convalescence, and are considered positive when the antibody titer in the convalescent phase increases by four or more times compared to the titer observed in the early phase of the disease. .

These methods cannot be used for early diagnosis of the disease and are not very suitable for establishing infective serotype due to cross reactions, especially in patients with heterologous immunity.

Due to the association between sequential infection with different serotypes and DHF / DSS, it is important to distinguish a primary infection from a secondary infection, with the HI test being the most used method for this purpose. Specific diagnosis of DENV infections can be made by viral isolation or molecular methods. Viral isolation is performed using blood obtained from patients who develop within 5 days after the onset of fever. Following isolation, viruses are identified and serotyped by immunofluorescence using serotype-specific monoclonal antibodies. Although the clinical sample is obtained in the acute phase of the disease, the final result of the cell culture can be up to 7 days, making early diagnosis impossible.

Currently, molecular methods have been used for the rapid diagnosis of dengue infections.

Recently, high levels of NS1 have been detected in the serum of patients in the acute phase of the disease using serological methods. Therefore, detection of this antigen has been routinely used in the main health centers.

Based on neutralization tests, DENV are classified into 4 immunologically distinct serotypes: DENV-1, DENV-2, DENV-3 and DENV-4. Genetic variants within each serotype have been identified and classified.

Neutralizing antibodies against DENV promote long-lasting immunity against reinfections of the same serotype, but not against infection with another serotype.

Primary DENV infections result in detectable IgM antibody levels, which persist for 1-2 months after infection.

IgM levels predominate over IgG levels at first infection, but in secondary infections IgG levels are similar to or greater than IgM levels at the onset of infection.

The produced IgM antibodies are directed against viral proteins E and NS1 and it has been observed that such antibodies can recognize NS1 protein from other serotypes.

In the first infection, the production of IgG antibodies starts from the convalescence phase and are directed to prM, E, NS1, NS3 and NS5 viral proteins.

DHF patients have an increase in IgG1 subclass antibodies when compared to patients with DF. Patients with PD, in turn, have increased antibodies to the IgG2 subclass. Several studies have shown the participation of CD4 + T cells and their mediators in cellular dengue immunity. The CD4 + T response is serotype specific, but these cells can also recognize other serotypes cross-over as they recognize several viral epitopes (C, E, NS3).

On the other hand, the participation of CD8 + T cells playing a direct cytolytic role has been demonstrated by recognizing specific serotype viral epitopes (E, NS1-2A, NS3) and can also recognize other serotypes in a cross-shape.

As already mentioned, in humans, dengue virus infection, regardless of serotype, produces permanent immunity against reinfection by the same serotype causing the infection, but not against other serotypes.

Epidemiological studies have shown that secondary infection (infection by a second serotype) with the presence of pre-existing antibodies against a different serotype is a risk factor for developing DHF / DSS. The absence of an adequate animal model makes it difficult to characterize the factors involved in viral pathogenesis. Host-dependent factors include age, genetic susceptibility, previous infection, and heterologous immunity.

Antibodies from previous infection do not neutralize another viral serotype and, on the contrary, facilitate both virion adsorption and entry into cells such as macrophages and monocytes. The virus within these cells evades phagolysosome degradation, increasing the rate of viral replication.

In vitro functional studies have shown that different viral genotypes present different characteristics of cellular infection, suggesting that virulence is related to the viral genotype.

Distinct DENV-2 strains isolated from patients with varying degrees of disease produce different levels of infectious virus in LLC-MK2 cells, peripheral blood leukocytes, and C6 / 36 cells. Also, several DENV-2 strains vary in their ability to infect the same cell type in vitro, depending on genotype and number of passages.

By sequencing the complete DENV-2 genome of Southeast Asian and American genotypes from the plasma of patients with FD and DHF / DSS, three regions of the genome (protein E, RNC 5 'and RNC 3') were identified. could be related to viral pathogenesis.

Infectious chimeric DENV-2 clones of Southeast Asian and American genotypes with these regions replaced showed a decrease in the release of macrophage virus and dendritic cells when the chimera has structures of the American genotype.

These results suggest that the protein coding region E, RNC 5 'and RNC 3' would have some influence on viral pathogenesis. Several studies suggest that viruses belonging to the same serotype, but of different genotypes, present differences in their pathogenesis. The difference in pathogenesis would be related to the structural conformation of viral proteins and non-coding regions. Identifying regions of the viral genome responsible for virulence would be very useful in epidemiological studies and may predict possible outbreaks of more severe cases and may even provide input for future vaccine preparation.

Thus, the present invention describes a process for constructing infectious RNAs from cDNAs of dengue virus type 3 (DENV-3) clones and chimeric DENV-3 and Rocio virus (ROCV) clones, as well as RNAs obtained. ROCV is arbovirus, a member of the Japanese Encephalitis Virus (JEV) serocomplex, and causes fatal encephalitis. Recently, an animal model to evaluate central nervous system neurovirulence caused by ROCV has been developed, but the molecular mechanisms that are related to viral factors have not yet been investigated.

Since glycoprotein E is the main component of the viral surface, responsible for phenotypic and antigenic properties, as well as for cellular tropism, the study of the participation of protein E in neurovirulence is of great importance.

Thus, the construction of an infectious clone is a useful tool that allows the manipulation of the viral genome to analyze possible genomic regions involved in the pathogenesis of DENV and ROCV, through in vivo and in vitro tests.

Prior art US 6,184,024 Bl describes the construction of recombinant flavivirus DNA using as the basic structure of the preparations the genome of dengue type 4 (strain 814669), a virus adapted in the laboratory after several passages in cell culture and / or brain of newborn mouse. The present invention utilizes a patient-isolated, non-laboratory adapted dengue type 3 virus with only few cell culture passages. In the prior art document several recombinant flavivirus DNAs have been constructed, but none include the Rocio virus. In addition, the vector plasmid into which recombinant DNAs were introduced and the RNA polymerase promoter for viral RNA transcription are different from those used in the present invention.

In MX 2010/007461 (A), reference is made to the construction of a DNA vaccine against dengue virus, which consists of a vector plasmid containing a part of the dengue virus genome type 1, 2, 3 and 4. individually or together. The present invention relates to the construction of recombinant DNA containing the entire viral genome.

In Brazilian document BR PI 0904020-0 (A2), the construction of recombinant DNA or DNA vaccine for protection against the four dengue virus serotypes is reported. It was prepared on the basis of the yellow fever virus vaccine strain genome, in which the virus E protein gene was replaced by the dengue virus protein E genes type 1, 2, 3 and 4, in a different strategy. proposed herein. US 2004/033594 (A1) describes an invention consisting of the construction of a recombinant DNA containing the tick-borne virus genome. The strategy used was based on restriction enzyme digestion and ligation of the different fragments until the whole viral genome was assembled.

In the invention described herein, artificial yeast chromosomes are used, in which assembly is performed by a process of gene recombination between overlapping genomic regions.

As can be seen, no prior art document describes the construction of infectious RNAs from DENV-3 and ROCV cDNAs, as well as said RNAs obtained, as proposed herein.

SUMMARY OF THE INVENTION: This invention describes processes for constructing infectious RNAs from cDNAs of dengue virus type 3 (DENV-3) clones and chimeric DENV-3 and Rocio virus (ROCV) clones, as well as the RNAs obtained. .

CDNAs represent the complete viral genome of Brazilian DENV-3 isolates, and DENV-3 / ROCV chimeric cDNAs are substituted for the DENV-3 protein E gene by the ROCV.

Brief Description of the Figures: Figure 1 schematically depicts the amplification of four DENV-3 (D3BR / RP1 / 2003) DNA fragments with ends homologous to the adjacent fragment. Figure 2 schematically represents the strategy of selecting colonies containing the complete DENV-3 cDNA inserted into the pYESIL vector. Figure 3 schematically depicts the amplification of DNA fragments with ends homologous to the adjacent fragment. Figure 4 schematically represents the fusion RT-PCR amplification strategy. Figure 5 schematically represents the strategy of selecting a colony containing the entire genome of chimeric viruses.

Detailed Description of the Invention: This invention describes methods for constructing infectious RNAs from cDNAs of dengue virus type 3 (DENV-3) clones and chimeric DENV-3 and Rocio virus (ROCV) clones.

To this end, two infectious clones of Brazilian isolates of DENV-3 (D3BR / RP1 / 2003 and D3BR / SL3 / 2002) and two chimeric ROCV viruses (SPH34675) were constructed.

In obtaining chimeric clones, protein E from isolated clones were replaced by ROCV protein E using the yeast cloning platform.

The infectious clones of dengue virus type 3 (DENV-3), strains D3BR / RP1 / 2003 and D3BR / SL3 / 2002, are listed in sequence listings no. 1 and 2, respectively.

Sequence listings related to the two chimeric viruses (D3BR / RP1 / 2003) / (SPH 34675) and (D3BR / SL3 / 2002) / (SPH 34675) are given in Seq Id. No. 3 and 4, respectively.

Following synthesis and transfection of viral RNAs, viral genomes were detected in the supernatant of C6 / 36 and Vero E6 cells, suggesting the infectious capacity of the constructs.

Isolates D3BR / RP1 / 2003 and D3BR / SL3 / 2002 (both with three passages in C6 / 36 cells) were chosen for amplification of overlapping cDNA fragments.

These isolates were selected because of their high similarity to most isolated DENV viruses when the nucleotide sequence of four genomic regions (RNC5 ', E, NS1 and RNC3') was analyzed. The SPH 34675 strain of the Rocio virus was prepared in the brain of newborn Swiss Mus musculus albino mice.

DENV-1, DENV-2 and DENV-4 viruses were amplified in newborn Swiss albino mouse (Mus musculus) brains.

Construction of a cDNA represents the genome. dengue virus type 3 integer (D3BR / RP1 / 2003 on D3BR / SL3 / 2002);

To construct the DENV-3 cDNA (strain D3BR / RP1 / 2003 or D3BR / SL3 / 2002), four overlapping, homologous-end cDNA fragments were amplified by RT-PCR and named A, B, C, and D as shown in Figure 1 and characterized in Table 1 below.

These fragments were mixed with the plasmid vector pYESIL and used to transform S. cerevisiae yeast (MaV203).

The four viral cDNA fragments were ligated and inserted into the pYESIL plasmid, directed by the homologous end sequences, by a gene recombination process.

To confirm the correct assembly of viral cDNA in the vector, several S. cerevislae colonies were analyzed by PCR using primers that flank the fragment recombination sites to obtain the diagnostic fragments (FD), as shown in Figure 2 and Table 2. below, down, beneath, underneath, downwards, downhill.

Colonies were considered positive when 569, 613, 1234, and 725 base pair fragments corresponding to the junctions between the A + B, B + C, C + D, D + vector fragments, respectively, were obtained.

Plasmids from several positive colonies were purified and subjected to viral cDNA nucleotide sequencing.

Plasmids containing the fewest mutations, when compared with the sequence of D3BR / RP1 / 2003 or D3BR / SL3 / 2002 strains, were selected and used to transform chemically competent E. coli cells.

A high transformation efficiency was observed, obtaining a large number of colonies. Several colonies were inoculated into liquid LB medium with 100 pg / ml spectinomycin antibiotic and, after 14 hours, the plasmids were purified showing an approximate size of 20 Kb as expected.

These plasmids were subjected to double digestion with the restriction enzymes Notl and Pad, which recognize sequences in the plasmid that flank the viral genome.

In double digestion, fragments of approximately 9.3 kb and 10.7 kb were observed, compatible with the size of the pYESIL vector and the viral genome, respectively.

These plasmids were subjected to nucleotide sequencing to confirm viral cDNA sequence.

Purified S. cerevisiae and E. coli plasmids were used for amplification of all viral cDNA by long PCR.

This cDNA was used as a template for viral RNA production by in vitro transcription. Subsequently, RNA was used to transfect C6 / 36 and Vero E6 cells.

After seven days of incubation, the viral genome was detected in the culture supernatant by real-time RT-PCR, as shown in Table 3 below.

Table 3: Real-time RT-PCR of RNA extracted from first pass cell supernatant. The process of constructing dengue virus type 3 cDNA (strain D3BR / RP1 / 2003 or strain D3BR / SL3 / 2002) is further detailed below. - Amplification of DNA fragments representing the entire genome of DENV-3, strain D3BR / RP1 / 2003 or strain D3BR / SL3 / 2002: The strategy used to introduce a cDNA representing the entire viral genome in the vector plasmid was to amplify, by polymerase chain reaction (PCR), four cDNA fragments (called A, B, C and D) with ends homologous to adjacent fragments, and the 5 '(A) and 3' (D) end fragments contain homologous regions with the pYESIL commercial vector.

The primers used were designed based on the genome sequence of the D3BR / RP1 / 2003 or D3BR / SL3 / 2002 isolates and the pYESIL vector and are shown in Table 4 below.

Table 4: Primers used for amplification of DNA fragments with homologous ends. ________________ Underlined sequences correspond to the pYESIL vector and bold sequences correspond to the T7 RNA polymerase promoter sequence. Fragment A was generated in two steps. The first step was performed using the AF-F and A-R primer pair. The AF-F primer contains the T7 RNA polymerase promoter sequence. In the second step, a 30 ng amplification product of the amplified product in the first step was used as a template and the pair of AF and AR primers finally generating fragment A, which has a 22 nucleotide insert at the 5 'end of the DNA, which represents the promoter sequence for T7 RNA Polymerase. PCR for fragment A was performed as follows: The reaction mixture, with a final volume of 50 ??, contained the following components: 30 ng of plasmid DNA and / or PCR product; 0.1 mM dNTP; 0.3 ?? of AF-F primer and 0.3 ?? A-R primer; 2.5 U enzyme DNA polymerase; 2 mM MgSO 4; 5 ?? of buffer comprising 600 mM Tris-SO4 and 180 mM ammonium sulfide. The reaction mixture was incubated at 94 ° C for 2 minutes, followed by 15 cycles of amplification at 94 ° C for 20 seconds, 45 ° C for 45 seconds and 68 ° C for 5 minutes.

Then the mixture is again exposed to 45 cycles at 94 ° C for 20 seconds, 56 ° C for 45 seconds, 68 ° C for 5 minutes, ending the reaction with an extension at 68 ° C for 10 minutes. Amplification of the BD fragments was performed using the same reaction mixture as indicated above, modifying only the primers and 45 amplification cycles as follows: 94 ° C for 20 seconds, 56 ° C for 45 seconds and 68 ° C for 5 minutes, finishing the reaction to a final extension at 68 ° C for 10 minutes.

An aliquot of 8 ?? of the amplification product was submitted to 1% agarose gel electrophoresis and subsequently the gel was stained and the bands were visualized with UV light.

The fragments were purified from agarose gel using an appropriate commercial kit according to the manufacturer's recommendations. - Assembly of the DENV-3 cDNA, strain D3BR / RP1 / 2003 or D3BR / SL3 / 2002, in S. cerevisiae: To construct the DENV-3 cDNA, a DNA fragment assembly system containing the pYESIL vector was used. and S. cerevisiae cells (MaV203). The pYESIL vector is an artificial yeast chromosome (YAC) that encodes the gene involved in tryptophan amino acid biosynthesis. In addition, it has an E. coli origin of replication and some unusual restriction enzyme dividing sites, such as Not I and Pad. · S. cerevisiae cells are unable to produce the essential amino acid tryptophan and can only grow in complete media (in the presence of this amino acid).

DNA fragments with ends homologous to adjacent fragments representing the entire viral genome (A-D) were mixed with the pYESIL vector and used to transform S. cerevisiae cells.

Transformed cells were seeded in solid medium plaques, called "tryptophan free amino acid" SC, and incubated at 30 ° C for 3 days.

Subsequently, a portion of the growing colonies were suspended at 15 ?? of the kit lysis buffer and a 1: 5 dilution of each colony was prepared and the suspension heated at 95 ° C for 5 minutes.

An aliquot of 0.5 ?? The cell lysate, previously heated to 95 ° C, was used to perform a colony PCR in an attempt to find a colony containing the assembled viral genome. PCR was performed using the diagnostic primers in Table 5 below.

Table 5: Primers used to amplify diagnostic fragments (FD) of DENV-3 cDNAs.

The primers used to generate the diagnostic fragments were designed based on the genomic sequence of Brazilian DENV-3 isolates and also based on the pYESIL vector.

The positive colonies were inoculated into 10 ml of tryptophan-free liquid "SC", incubated at 30 ° C and shaken at 150 rpm for 2 days.

A 1.5 ml aliquot of the culture from each colony was used for plasmid DNA extraction using a specific commercial kit. - Transformation of competent E. coli cells with plasmid containing viral genome: Plasmids extracted from the S. cerevisiae culture were used to transform E. coli cells.

Approximately 50 ng of plasmid DNA was added to 50 ?? of competent cells and this mixture was incubated on ice for 30 minutes.

Subsequently, the cells were subjected to thermal shock (42 ° C, 45 seconds). After 5 minutes incubation on ice, 250 µl were added to the tube. of SOC medium. Cells were incubated at 37 ° C with shaking at 200 rpm for 2 hours and then seeded in LB-agar-containing Petri dishes containing 100 pg / ml spectinomycin antibiotic incubated at 37 ° C for 14 hours.

Subsequently, several colonies were inoculated into 6 ml of liquid LB medium containing 100 pg of the spectinomycin antibiotic and incubated at 37 ° C at 200 rpm for 14 hours. To verify that the insert size is compatible with the expected size, approximately 2 pg of the plasmid DNAs were digested with the restriction enzymes Notl and Pad.

The colonies analyzed were stored at -80 ° C, with 15% glycerol added and 5 ml of each colony culture was subjected to plasmid purification using an appropriate kit. - Nucleotide Tracking: Plasmids obtained from S. cerevisiae and E. coli were subjected to nucleotide sequencing using a commercial sequencing kit following the protocol recommended by the manufacturer.

Following the sequencing reaction, the labeled DNA product was sequenced in automated sequencer. - Viral genome amplification by long PCR: The plasmids obtained from S. cerevisiae and E. coll were used to perform a long PCR for viral genome amplification. The reaction mixture contained 3 pL (about 30 ng), the AF-F and RNC-3 'primers (D1, D2, D3), described in Table 7 below.

Table 7: Primers used for DENV-3 whole genome sequencing.

The reagent concentrations of this reaction are the same as mentioned above. The mixture is heated at 94 ° C for 2 minutes, followed by 60 cycles of 94 ° C for 20 seconds, 62 ° C for 45 seconds, 68 ° C for 11 minutes; followed by a final extension of 11 minutes at 68 ° C.

Subsequently, an approximately 10,707 bp fragment corresponding to the entire cDNA genome was purified. - In vitro transcription of viral cDNA: A purified 10.7 Kb fragment (about 1 pg) t corresponding to the entire viral genome was used for in vitro transcription of viral RNA using T7 RNA Polymerase for 8 hours, following protocol recommended by manufacturer. The cDNA was digested with a DNasel by. 15 minutes, incubated at room temperature and subsequently viral RNA was purified with appropriate commercial kit following manufacturer's specifications. - Viral RNA Transfection: A mixture called A containing 1 ?? of transfection reagent and 49 ?? medium without SFB (fetal bovine serum) and antibiotic, and another mixture called B containing 25 ?? of viral RNA (about 0.5-1 µg) and 25 ?? medium without SFB and antibiotic were incubated for 5 minutes at room temperature.

Subsequently, mixtures A and B were incubated together for 20 minutes at room temperature and after that period were used to transfect cells simulating a viral infection.

After 1 hour 900 were added ?? of culture medium L15 and C6 / 36 and Vero cells were incubated at 28 or 37 ° C, respectively.

After 4 hours, the culture media was replaced with 2% SFB and antibiotic media. Between 7-10 days after transfection, cells and supernatant were collected for detection of the viral genome by real-time RT-PCR. - Real-time RT-PCR to quantify viral load: Has viral RNA been purified from 140? C6 / 36 culture supernatant using appropriate kit following manufacturer's recommended protocol. The viral RNA was then suspended at 80 ?? RPC free DEPC water pump. Real-time RT-PCR was performed as previously described. The viral construct obtained is useful as a tool for the study of pathogenesis related aspects, such as molecular determinants involved in virulence, and may also provide subsidies for future vaccines. The entire cDNA can also serve as template DNA for generating a transcribed viral RNA to act on positive control in different genomic detection methods.

Construction of a Cryptic Dengue Virus Type 3 (D3BR / RP1 / 2003 or D3BR / SL3 / 2002) and Rocio (SPH 34675) cDNA To construct a DENV-3 / ROCV chimeric cDNA, the DENV-3 protein E gene (D3BR / RP1 / 2003 or D3BR / SL3 / 2002) was replaced by the ROCV protein E gene (SPH 34675).

To achieve this goal, four overlapping, homologous end fragments were PCR amplified and named AQ, BQ, CQ, and DQ, as shown in Figure 3 and characterized in Table 8 below.

Table 8: Amplified cDNA Fragments The AQ fragment was obtained by fusion of the AF + BF fragments, thus generating the fragment called AQF. The BF fragment contains the ROCV protein E gene, as shown in Figure 4, and the AQF fragment was used as a template to generate the AQ fragment, as shown in Figure 4.

These fragments were mixed with the plasmid vector pYESIL and used to transform S. cerevisiae yeast (MaV203).

The four viral cDNA fragments were ligated and inserted into the pYESIL plasmid, directed by the homologous end sequences, by a gene recombination process.

To confirm the correct assembly of viral cDNA in the vector, several S. cerevisiae colonies were analyzed by PCR using primers that flank the fragment recombination sites to obtain the diagnostic fragments (FD), as shown in Figure 5 and Table 9. below, down, beneath, underneath, downwards, downhill.

Table 9: Diagnostic Fragments _____________ Size Fragments (bp) Genome Position Colonies were considered positive when fragments of 569, 613, 516, and 725 base pairs corresponding to the junctions between AQ + BQ, BQ + CQ, CQ + DQ fragments , DQ + vector, respectively, were obtained.

Plasmids from several positive colonies were purified and subjected to viral cDNA nucleotide sequencing.

Plasmids containing the smallest number of mutations, when compared to the sequence of the D3BR / RP1 / 2003 or D3BR / SL3 / 2 02 strain, were selected and used to transform chemically competent E. coli cells.

A high transformation efficiency was observed, obtaining a large number of colonies. Several colonies were inoculated into liquid LB medium with 100 pg / ml spectinomycin antibiotic and, after 14 hours, the plasmids were purified showing an approximate size of 20 Kb as expected.

These plasmids were subjected to double digestion with the restriction enzymes Notl and Pad, which recognize sequences in the plasmid that flank the viral genome.

In double digestion, fragments of approximately 9.3 kb and 10.7 kb were observed, compatible with the size of the pYESIL vector and the viral genome, respectively.

These plasmids were subjected to nucleotide sequencing to confirm viral cDNA sequence.

Purified S. cerevisiae and E. coli plasmids were used as a template for amplification of all viral cDNA by long PCR.

This cDNA was used as a template for viral RNA production by in vitro transcription. Subsequently, RNA was used to transfect C6 / 36 and Vero E6 cells.

After seven days of incubation, the viral genome was detected in the culture supernatant by real-time RT-PCR, as shown in Table 10 below.

Table 10: Real-time RT-PCR of RNA extracted from first pass cell supernatant. The chimeric cDNA construction process of dengue virus type 3 (D3BR / RP1 / 2003 and D3BR / SL3 / 2002) and Rocio ( SPH 34675) is further detailed below. - Extraction of viral RNA: Has viral RNA been purified from 140? C6 / 36 culture supernatant using appropriate commercial kit following manufacturer's recommended protocol. The viral RNA was then suspended at 80 ?? RPC free DEPC water pump. - Reverse Transcription (RT): The reaction mixture for cDNA synthesis (total volume 40 ??) has been added: 24 ?? RNA; 100 ng of random primers; 0.125 mM dNTP including 8 ?? buffer containing 250 mM Tris-HCL, 375 mM KCl and 15 mM MgCl2 / 80 U RNAse inhibitor and 400 U reverse transcriptase. The mixture was incubated at 25 ° C for 10 minutes, followed by an incubation at 37 ° C for 4 hours and finally 5 minutes at 85 ° C.

Subsequently, cDNAs were treated with 2 U RNAse and incubated at 37 ° C for 30 minutes, stored at -20 C. - Amplification of DNA fragments representing chimeric dengue virus type 3 cDNA (D3BR / RP1 / 2003 or D3BR / SL3 / 2002) / Rocio (SPH 34675): The strategy used to introduce a cDNA representing the chimeric cDNA in which the D3BR / RP1 / 2003 or D3BR / SL3 / 2002 strain DENV-3 protein E gene is replaced by the ROCV protein E gene from strain SPH 34675 was amplified by fusion PCR.

The primers used were designed based on the genome sequence of isolates D3BR / RP1 / 2003 and D3BR / SL3 / 2002, ROCV SPH 34675 and the pYESIL vector and are shown in Table 11 below.

Table 11: Primers used for amplification of DNA fragments with homologous ends. __________ Initially, a fusion PCR was used as described below.

A DNA fragment from DENV-3 (AF fragment) including nucleotides 1 through 934 was PCR amplified using the AF-F sense primer, which contains at its 5 * end the RNA polymerase promoter sequence and the primer. Complementary AF-R, which corresponds to the last sequences of the prM protein gene. The ROCV protein E gene (BF fragment) was amplified using the BF-F sense primer, which contains at its 5 'end the sequence corresponding to the last nucleotides of the DENV-3 prM protein gene and the BF primer -R complementary, which contains at its 3 'end the initial sequence of the DENV-3 NS1 protein gene. The PCR reaction mixture, with a final volume of 50 ??, contained the following components: 30 ng of plasmid DNA or 5 ?? cDNA; 0.1 mM dNTP; 0.3 ?? of forward (sense) primers and 0.3 ?? reverse primers; 2.5 U enzyme DNA polymerase; 2 mM MgSO 4; 5 ?? respective buffer 600 mM Tris-SC> 4, 180 mM ammonium sulfide.

The cycle temperatures for generating the AF and BF fragment was 94 ° C for 2 minutes, followed by 15 amplification cycles as follows: a) 94 ° C for 20 seconds, b) 45 ° C for 45 seconds and c) 68 ° C for 5 minutes for extension; followed again by 45 cycles of: a) 94 ° C for 20 seconds, b) 56 ° C for 45 seconds, c) 68 ° C for 5 minutes, terminating the reaction with a final extension at 68 ° C for 10 minutes.

An aliquot of 8 ?? of the amplification product was submitted to 1% agarose gel electrophoresis. Subsequently, the gel was stained and the bands were visualized with UV light.

The fragments were purified from the agarose gel using an appropriate extraction kit following the manufacturer's recommendations.

These fragments were subjected to fusion PCR to generate the AQF fragment. The reaction mixture contained the same components as above, but using approximately 30 ng of each of the AF and BF fragments, and the AF-F and BF-R primers using the amplification cycles mentioned above. The AQF fragment was purified from the gel as mentioned above. This fragment was used as a template in a PCR with AQ-F primer, which has at its 5 'end a sequence homologous to the pYESIL vector sequence and the AQ-R primer to generate the AQ fragment, following the protocol mentioned above.

Three other fragments were generated by PCR (BQ, CQ and DQ) and amplified using the same reagent concentrations as the above-mentioned PCR protocol.

The temperatures for amplifying these fragments were: a) 94 ° C for 20 seconds for denaturation, b) 56 ° C for 45 seconds for primer binding and c) 68 ° C for 5 minutes for extension and terminating the reaction with a final extension to 68 ° C for 10 minutes. - Assembly of the whole cfuimeric dengue virus type 3 cDNA (D3BR / RP1 / 2 003 or D3BR / SL3 / 2002) / Rocio (SPH 34675) in S. cerevisiae: For the construction of the DENV-3 chimeric cDNA (D3BR / SRP1) / 2003 or D3BR / SL3 / 2002) / Rocio (SPH 34675), a DNA fragment assembly system containing the pYES1L vector and S. cerevisiae cells (MaV203) was used. The pYESlL vector is an artificial yeast chromosome (YAC) that encodes the gene involved in the tryptophan amino acid biosynthesis. In addition, it has an E. coli origin of replication and some unusual restriction enzyme dividing sites, such as Notl and Pad.

S. cerevisiae cells are unable to produce the essential amino acid tryptophan and can only grow in complete media (in the presence of this amino acid).

DNA fragments with ends homologous to adjacent fragments representing the entire viral genome were mixed with the pYES1L vector and used to transform S. cerevisiae cells.

Transformed cells were seeded in solid medium plaques, called "tryptophan free amino acid" SC, and incubated at 30 ° C for 3 days.

Subsequently, a portion of the growing colonies were suspended at 15 ?? of the kit lysis buffer and a 1: 5 dilution of each colony was prepared and the suspension heated at 95 ° C for 5 minutes.

An aliquot of 0.5 ?? The cell lysate, previously heated to 95 ° C, was used to perform a colony PCR in an attempt to find a colony containing the assembled viral genome. PCR was performed using the diagnostic primers from Table 12 below.

Table 12: Primers used to amplify the diagnostic fragments (FD) of the chimeric clones ._____________ The primers used to generate the diagnostic fragments were designed based on the genomic sequences of the Brazilian DENV-3 isolates (D3BR / RP1 / 2003 and D3BR / SL3 / 2002) and ROCV (SPH 34675) and also based on the pYESlL vector.

The positive colonies were inoculated into 10 ml of tryptophan-free liquid "SC", incubated at 30 ° C and shaken at 150 rpm for 2 days.

A 1.5 ml aliquot of the culture from each colony was used to extract DNA containing the viral genome using a specific commercial kit. - Transformation of competent cells: Plasmids extracted from the S. cerevisiae culture were used to transform E. coli cells.

Approximately 50 ng of plasmid DNA was added to 50 ?? of competent cells and this mixture was incubated on ice for 30 minutes.

Subsequently, the cells were subjected to thermal shock (42 ° C, 45 seconds). After 5 minutes incubation on ice, 250 µl were added to the tube. of SOC medium. Cells were incubated at 37 ° C with shaking at 200 rpm for 2 hours and then seeded in LB-agar-containing Petri dishes containing 100 pg / ml spectinomycin antibiotic incubated at 37 ° C for 14 hours.

Subsequently, several colonies were inoculated into 6 ml of liquid LB medium containing 100 pg of the spectinomycin antibiotic and incubated at 37 ° C at 200 rpm for 14 hours.

To verify that the insert size is compatible with the expected size, approximately 2 pg of plasmid DNAs were digested with restriction enzymes Notl and FacI.

The colonies analyzed were stored at -80 ° C, with 15% glycerol added and 5 ml of each colony culture was subjected to plasmid purification using an appropriate kit. - Nucleotide sequencing: Plasmids obtained from S. cerevisiae and E. coli were subjected to nucleotide sequencing using a commercial sequencing kit following the protocol recommended by the manufacturer.

Following the sequencing reaction, the labeled DNA product was sequenced in automated sequencer.

- Viral genome amplification by long PCR: Plasmids obtained from S. cerevisiae and E. coli were used to perform a long PCR for viral genome amplification. The reaction mixture contained 3pl (about 30 ng), the AF-F and RNC-3 'primer (D1, D2, D3), already described in Table 7.

The reagent concentrations of this reaction are the same as mentioned above. The mixture is heated at 94 ° C for 2 minutes, followed by 60 cycles of 94 ° C for 20 seconds, 62 ° C for 45 seconds, 68 ° C for 11 minutes; followed by a final extension of 11 minutes at 68 ° C.

Subsequently, an approximately 10,707 bp fragment corresponding to the entire cDNA genome was purified. - In vitro transcription of viral cDNA: A purified 10.7 kb fragment (about 1 pg), corresponding to the entire viral genome, was used for in vitro transcription of viral RNA using RNA Polymerase for 8 hours, following protocol recommended by the manufacturer. . The cDNA was digested with a DNasel for 15 minutes, incubated at room temperature and subsequently the viral RNA was purified with an appropriate commercial kit following the manufacturer's specifications. - Viral RNA Transfection: A mixture called A containing 1 ?? of transfection reagent and 49 ?? medium without SFB (fetal bovine serum) and antibiotic, and another mixture called B containing 25 ?? of viral RNA (about 0.5-1 pg) and 25 ?? medium without SFB and antibiotic were incubated for 5 minutes at room temperature.

Subsequently, mixtures A and B were incubated together for 20 minutes at room temperature and after that period were used to transfect cells simulating a viral infection.

After 1 hour 900 were added ?? of culture medium L15 and C6 / 36 and Vero cells were incubated at 28 or 37 ° C, respectively.

After 4 hours, the culture media was replaced with 2% SFB and antibiotic media. Between 7-10 days after transfection, cells and supernatant were collected for detection of the viral genome by real-time RT-PCR. - Real-time RT-PCR to quantify viral load: Has viral RNA been purified from 140? C6 / 36 culture supernatant using appropriate kit following manufacturer's recommended protocol. The viral RNA was then suspended at 80 ?? RPC free DEPC water pump. Real-time RT-PCR was performed as previously described. The obtained chimeric construction is useful as a tool for the study of pathogenesis related aspects, such as molecular determinants involved in Rocio virus neuroinvasivity and neurovirulence, and may also provide subsidies for future vaccines. Chimeric cDNA can also serve as template DNA for generating a transcribed viral RNA to act on positive control in different genomic detection methods.

Claims (20)

1. Process for the construction of infectious RNAs from cDNAs of dengue virus type 3 strains D3BR / RP1 / 2003 and D3BR / SL3 / 2002, characterized by the steps of: A) amplification of four fragments cDNA with overlapping and homologous ends by RT-PCR; B) mixing the fragments with plasmid vector pYESlL and transforming S. cerevisiae yeast (MaV203); C) selection, purification and nucleotide sequencing of plasmids containing the fewest mutations; D) transformation of chemically competent E. coli cells by the selected plasmids and further purification and nucleotide sequencing; E) amplification of all viral cDNA by long PCR using purified S. cerevisiae and E. coli plasmids; F) production of viral RNA from cDNA by in vitro transcription; and G) transfection of C6 / 36 and Vero E6 cells. Process according to Claim 1, characterized in that, in step (A), amplification is promoted by the AF-F, AF, AR, BF, BR, CF, CR, DF and DR primers in Seq Ids. No. 5 to 13. Process according to Claim 1 or 2, characterized in that, in step (A), the four amplified fragments have 3024 bp, 2839 bp, 3028 bp and 1930 bp respectively, being found in the genome in the positions 1 - 3024, 2986 - 5825, 5787 - 8815 and 8777 - 10707. Process according to claim 1 or 2, characterized in that, in step (A), the fragments were subjected to 1% agarose gel electrophoresis and further purification. Process according to Claim 1 or 2, characterized in that, in step (B), S. cerevisiae cells are transformed and seeded in tryptophan-free "SC" medium and incubated at 30 ° C for 3 hours. days for subsequent suspension and PCR analysis. Process according to claim 1, characterized in that, in step (C), the primers used to generate the diagnostic fragments of S. cerevisiae colonies containing the plasmids with the assembled cDNAs are: PD (A + B) -F, PD (A + B) -R, PD (B + C) -F, PD (B + C) -R, PD (C + D) -F, PD (C + D) -R , PD (D + vector) -F and PD (D + vector) -R, presented in Id. No. 14 to 21. Process according to Claim 1, characterized in that, in step (D), the plasmids were subjected to double digestion with the restriction enzymes Not I and Pad. Process according to Claim 1, characterized in that, in step (E), amplification is promoted by the AF-F primers, presented in Id. De Seq. No. 5, and RNC-3 '(D1, D2, D3). Process according to Claim 1, characterized in that, at the end of the process, viral RNA is analyzed by real-time RT-PCR to quantify its viral load. 10. cDNA of clones of dengue virus type 3 strains D3BR / RP1 / 2003 and D3BR / SL3 / 2002 characterized by the fact that they are presented in the Id. Of Seq. 1 and 2. 11. Process for the construction of infectious RNAs from cDNAs of chimeric clones of D3BR / RP1 / 2003 and D3BR / SL3 / 2002 dengue virus type 3 strains and Rocio virus SPH 34675 strain, characterized by the fact that it comprises the stages of: A) replacement of the dengue virus type 3 protein E gene by the Rocio virus protein E gene; B) amplification of four homologous overlapping cDNA fragments by fusion PCR; C) mixing the fragments with the pYESIL plasmid vector and transforming the yeast S. cerevisiae (MaV203); D) selection, purification and nucleotide sequencing of plasmids containing the fewest mutations; E) transformation of chemically competent E. coli cells by the selected plasmids and further purification and nucleotide sequencing; F) amplification of all viral cDNA by long PCR using purified S. cerevisiae and E. coli plasmids; G) production of viral RNA from cDNA by in vitro transcription; and H) transfection of C6 / 36 and Vero E6 cells. Process according to Claim 11, characterized in that, in step (B), amplification is promoted by the primers AQ-F, AQ-R, BQ-F, BQ-R, CQ-F, CQ. -R, DQ-F and DQ-R, shown in Seq. No 26 to 33. Process according to Claim 11, characterized in that, in step (B), the four amplified fragments have, respectively, 2500 bp, 3201 bp, 3028 bp and 2121 bp, being found in the genome at positions 1 - 2428, 2414 - 5615, 5586 - 8624 and 8586 - 10707. Process according to Claim 11 or 12, characterized in that, in step (B), the fragments were subjected to 1% agarose gel electrophoresis and further purification. Process according to Claim 11, characterized in that, in step (C), the primers used to generate the diagnostic fragments of S. cerevisiae colonies containing the plasmids with the assembled cDNAs are: PD (A + B) -F / 1, PD (A + B) -F, PD (A + B) -R, PD (B + C) -F, PD (B + C) -R, PD (C + D) -F, PD (C + D) -R, PD (D + vector) -F and PD (D + vector) -R, shown in Id. 34 and 14 to 21. Process according to Claim 11, characterized in that, in step (C), S. cerevisiae cells (MaV203) are transformed and seeded in tryptophan-free "SC" medium and incubated at 30 ° C for 30 minutes. 3 days for subsequent suspension and PCR analysis. Process according to Claim 11, characterized in that, in step (D), the complete protein E gene sequencing of chimeric cDNAs is promoted by the RocV-Env-1F, RocV-Env-2F primers, RocV-Env-3F and RocV-Env-4F, shown in Seq. No. 22 to 25 Process according to Claim 11, characterized in that, in step (E), the plasmids were subjected to double digestion with restriction enzymes Not I and Pad. Process according to Claim 11, characterized in that at the end of the process viral RNA is analyzed by real-time RT-PCR for quantification of its viral load. 20. cDNA of chimeric clones of dengue virus type 3 strains D3BR / RP1 / 2003 and D3BR / SL3 / 2002 and Rocio virus strain SPH 34675 characterized by the fact that they are presented in Seq Id. No. 3 and 4.