Searching for the best real-time RT-PCRs to detect Zika virus infections: the importance of comparing several protocols.

Clinical manifestations of Zika, dengue, and chikungunya virus infections are very similar, making it difficult to reach a diagnosis based only on clinical grounds. In addition, there is an intense cross-reactivity between antibodies directed to Zika virus and other flaviviruses, and an accurate Zika diagnosis is best achieved by real-time RT-PCR. However, some real-time RT-PCR show better performance than others. To reach the best possible Zika diagnosis, the analytic sensitivity of some probe-based real-time RT-PCR amplifying Zika virus RNA was evaluated in spiked and clinical samples. We evaluated primers and probes to detect Zika virus, which had been published before, and tested sensitivity using serum spiked and patient samples by real-time RT-PCR. When tested against spiked samples, the previously described primers showed different sensitivity, with very similar results when samples from patients (serum and urine) were analyzed. Real-time RT-PCR designed to amplify Zika virus NS1 showed the best analytical sensitivity for all samples.

Comparison of techniques for extracting viral RNA from isolation-negative serum for dengue diagnosis by the polymerase chain reaction.

Aiming at the improvement of the molecular diagnosis of dengue, three well-established methods of RNA extraction from serum of patients with clinical symptoms of dengue were compared. The methods were based on the QIAamp Viral RNA kit, the Chomczynski-Sacchi technique and TRIzol. One hundred samples were examined using the same protocol for reverse transcription-polymerase chain reaction (RT-PCR). Out of the 100 samples tested, none was positive by either the Chomczynski-Sacchi technique or TRIzol, and six were positive using the QIAamp viral RNA kit. Of the six positive samples, only one was collected before 5 days of the beginning of the disease, and it was also positive for viral isolation. These results were confirmed later by serology (MAC-ELISA) that showed that 19 samples were positive for IgM antibodies against dengue. These data indicate that PCR is a useful method for detection of dengue virus infections in IgM-positive samples, and the best method of RNA extraction from clinical samples, to be used for dengue diagnosis by PCR is the QIAamp Viral RNA kit.

Recombinant plasmid expressing a truncated dengue-2 virus E protein without co-expression of prM protein induces partial protection in mice.

A nucleic acid vaccine candidate against dengue-2 virus was constructed to express a truncated dengue-2 E glycoprotein without concomitant expression of prM. The truncated E protein was properly expressed even in the absence of prM. Mice inoculated intramuscularly with the recombinant plasmid containing 94% of the E gene did not respond with anti-dengue antibodies, cellular proliferation, or synthesis of cytokines by their lymphoid cells when stimulated with purified dengue-2 virus. However, protection was observed in 20% of the challenged mice immunized with this recombinant plasmid and the mice survived longer than the control group. The low percentage of protection might be explained by a weak activation of the immune system resulting from an imperfect secretion of E due to lack of the prM protein. This study corroborates with the hypothesis that prM is important for the processing of the E glycoprotein and should be incorporated on candidate vaccines engineered by recombinant DNA technology.