**3. Results**

## *3.1. Demographic and Clinical Data*

During the study period from September 2015 to March 2016, 104 children aged between 1 and 115 months were enrolled and screened for DENV, CHIKV, ZIKV, YFV, RVFV—Seventy-nine were <5 years old, and 42 (40,38%) were male (Supplementary Data 1). Regarding clinical symptoms, the most common symptoms after fever were, respectively, rhinorrhea (65%), cough (53%; 55/104), vomiting (25%; 26/104), diarrhoea (25%; 25/104) and abdominal pain (25%; 26/104). Headaches were reported only in 10% of the enrolled patients, while myalgia and arthralgia were not recorded at all (Figure 3).

**Figure 3.** Plot showing the percentage of recorded symptoms among enrolled patients.

## *3.2. Molecular, Serologic and Antigenic Detection*

Three samples (2.8%) tested positive with DENV1-3 RPA, while none of the other arbovirus assays yielded positive results. All of the arbovirus results were confirmed by RT-PCR, with 100% concordance. The samples of the three DENV cases were additionally tested and confirmed positive by DENV NS1 antigen capture (Table 1). The ELISA IgM test yielded negative results in all three cases (not shown). Virus isolation at 28 ◦C in C6/36 cells yielded non-obvious CPE ten days after inoculation. However, two strains were successfully isolated, and isolation was confirmed by IFA (Figure S1). The isolates were designated Medina Gounass 1 and Medina Gounass 2, respectively.


**Table 1.** Comparison of time detection between non-structural protein 1 (NS1), real-time recombinase polymerase amplification (RT-RPA), and RT-PCR.

## *3.3. Phylogenetic Analysis*

Finally, the sequence of the CprM gene of the two isolates was determined and deposited in genbank (accessions numbers MK940790 (Medina gounass 1/2015), and MK940791 (Medina gounass 1/2015)). Both strains were completely homologous (100%), with no nucleotide difference. A basic local alignment search tool for nucleotide (BLASTN), using the obtained C-prM gene sequence, showed 100% identity with the DENV-1 isolates collected in Guangzhou, China, in 2014 (KP279753.1). A calculated phylogenetic tree clustered the determined DENV-1 sequences with Asian strains, supported by high bootstrap values (Figure 4).

**Figure 4.** Phylogenetic tree based on the C-prM gene sequences using the maximum likelihood (ML) method, showing the relationship of 2 from 3 isolated viruses in this study (darkred circle) with 40 global samples of dengue virus (DENV), and Kimura 2 parameters; the gamma distributed (K2+G+I) nucleotide substitution model was used. The yellow fever 17D (FJ654700.1) was used as the outgroup.
