Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

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.

Development, verification, and validation of an RT-qPCR-based protocol for Yellow Fever diagnosis.

INTRODUCTION: Yellow fever (YF) is a public health threat with frequent outbreaks in tropical and subtropical areas, despite the existence of a safe and effective vaccine. The diagnosis of acute infection of the etiologic agent relies mainly on real-time reverse transcription-polymerase chain reaction (RT-qPCR)-based assays. OBJECTIVES: The aim of this study was to evaluate and compare this novel protocol for yellow fever virus (YFV) diagnosis against assays developed in-house by reference laboratories for arboviruses. METHODS: We developed a novel molecular protocol for the detection of YFV that includes an Internal Control to validate the reaction and an External Control to monitor the RNA extraction efficiency. RESULTS AND DISCUSSION: Our assay detects one viral genome per reaction and displays no cross-reactions with dengue (1-4), Zika, or Chikungunya viruses. This novel assay yielded 95% of agreement with the reference method recommended by the Pan American Health Organization when analyzing 204 clinical samples and cultured viruses, these samples were analyzed in 3 different diagnosis centers for arboviruses in Brazil. The data suggest the use of the proposed multiplex assay protocol to do routine tests in a clinical laboratory. This product adds higher specificity and sensitivity in addition to reduced cost per test due to hands-on time and reagent spending.

Phylogenetic Characterization of Arboviruses in Patients Suffering from Acute Fever in Rondônia, Brazil.

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.

External Control Viral-Like Particle Construction for Detection of Emergent Arboviruses by Real-Time Reverse-Transcription PCR.

Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1-4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotypes 1-4, chikungunya, and an internal human control. RNA extracted from the external control was able to be amplified and detected in RT-qPCR assays for each virus detected by using the ZDC Biomol kit. The external control, samples from viral culture, and infected patient samples display similar amplification using this assay. The pET47b(+)MS2-ZDC vector is a viable expression system for the production of external control viral-like particles (MS2-ZDC). The RNA from the recombinant particles can be easily extracted and can function as a tool to validate all steps of process from the extraction to the amplification of all targets in specific reaction. Thus, the MS2-ZDC particles are laboratory-safe in order to avoid risk for operators, and the phages are effective as positive control for use in the ZDC Biomol kit amplifying all kit targets making them effective for commercial profile.

Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil.

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.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

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.