Several arboviruses have emerged and/or re-emerged in North, Central and South-American countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list.

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test.OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses.METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection.FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

The purpose of the study was to classify, through phylogenetic analyses, the main arboviruses that have been isolated in the metropolitan region of Porto Velho, Rondônia, Brazil. Serum samples from patients with symptoms suggesting arboviruses were collected and tested by One Step RT-qPCR for Zika, Dengue (serotypes 1–4), Chikungunya, Mayaro and Oropouche viruses. Positive samples were amplified by conventional PCR and sequenced utilizing the Sanger method. The obtained sequences were aligned, and an evolutionary analysis was carried out using Bayesian inference. A total of 308 samples were tested. Of this total, 20 had a detectable viral load for Dengue, being detected DENV1 (18/20), co-infection DENV1 and DENV2 (1/20) and DENV4 (1/20). For Dengue serotype 3 and for the CHIKV, ZIKV, MAYV and OROV viruses, no individuals with a detectable viral load were found. A total of 9 of these samples were magnified by conventional PCR for sequencing. Of these, 6 were successfully sequenced and, according to the evolutionary profile, 5 corresponded to serotype DENV-1 genotype V, and 1 to serotype DENV-4 genotype II. In the study, we demonstrate co-circulation of the DENV-1 genotype V and the DENV-4 genotype II. Co-circulation of several DENV serotypes in the same city poses a risk to the population and is correlated with the increase of the most severe forms of the disease. Similarly, co-circulation of genetically distinct DENV and the occurrence of simultaneous infections can affect recombination events and lead to the emergence of more virulent isolates.

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