A 57 year old Portuguese man, with no past medical history, presented on December 7 2015 complaining of chills, generalized asthenia and myalgia and rash, one day after returning from Rio de Janeiro after a 10 day stay. The study in the hospital revealed thrombocytopenia (126 000/muL), without leukopenia or abnormal liver enzymology. Real-time PCR was positive for Zika virus in the urine sample.
The patient received supportive treatment, improving clinically to symptoms free.
Previous flavivirus infections and vaccination against flaviviruses (yellow fever, tick-borne encephalitis and Japanese encephalitis viruses) were not reported by any of the patients. The data of these four imported cases to Portugal is summarized in Table 1.
In the National Institute of Health, sera samples were tested by immunofluorescent assay (IFA) in-house (IgG and IgM) for Zika virus (ZIKV), Dengue (DENV) and Chikungunya viruses (CHIKV) (Table 1). The presence of immunoglobulins specific to other flaviviruses, such as yellow fever (YFV), tick-borne encephalitis (TBEV) and West Nile (WNV) viruses was also tested by IFA and all assay results were negative for IgM and presented cross-reaction signals in IgG.
Nucleic acids were extracted from 400 muL of ethylenediaminetetraacetic acid (EDTA) blood samples and 900 muL urine samples using NucliSens easyMAG platform (BioMerieux). The presence of ZIKV RNA was checked by real-time RT-PCR and conventional reverse transcription-polymerase chain reaction (RT-PCR) using Zika specific primers and pan-flavivirus primers targeting the non-structural protein 5 (NS5) gene. The presence of DENV and CHIKV RNA was checked by real-time PCR as reported previously, and by conventional RT-PCR, respectively. For cases 1 and 2, Zika RT-PCR was negative (conventional and real time RT-PCR) on blood samples collected on day 13 post onset of disease. For cases 3 and 4, real time RT-PCR and conventional RT-PCR were positive in urine samples on day 8 and 9 post onset, respectively. The nested- PCR fragments (overlapping NS5, 205 bp) was sequenced bi-directionally and submitted to GenBank (accession numbers KU752544 and KU752545). Similarity searches were made within the GenBank data set using the basic local alignment search tool (BLAST) BLASTN algorithm. Maximum likelihood analysis was performed with Kimura two-parameter model with invariant sites using Mega version 6 software. The robustness of the nodes was tested by 1000 bootstrap replications.