A bstract: Background: Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology–which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa. Results: The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes. Conclusion: We have developed a rapid, sensitive and specific rRT – PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection.

The human MxA gene belongs to the class of interferon (IFN)-stimulated genes (ISGs) involved in antiviral resistance against influenza viruses. Here, we studied the requirements for MxA induction by influenza A virus infection. MxA is transcriptionally upregulated by type I (alpha and beta) and type III (lambda) IFNs. Therefore, MxA is widely used in gene expression studies as a reliable marker for IFN bioactivity. It is not known, however, whether viruses can directly activate MxA expression in the absence of secreted IFN. By using an NS1-deficient influenza A virus and human cells with defects in IFN production or the STAT1 gene, we studied the induction profile of MxA by real-time reverse transcriptase PCR. The NS1-deficient virus is known to be a strong activator of the IFN system because NS1 acts as a viral IFN-antagonistic protein. Nevertheless, MxA gene expression was not inducible by this virus upon infection of IFN nonproducer cells and STAT1-null cells. Likewise, neither IFN-α nor IFN-λ had a sizeable effect on the STAT1-null cells, indicating that MxA expression requires STAT1 signaling and cannot be triggered directly by virus infection. In contrast, the expression of the IFN-stimulated gene ISG56 was induced by influenza virus in these cells, confirming that ISG56 differs from MxA in being directly inducible by viral triggers in an IFN-independent way. In summary, our study reveals that MxA is a unique marker for the detection of type I and type III IFN activity during virus infections and IFN therapy.

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4–10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection.

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.