In Jazan region of southwestern Saudi Arabia, several outbreaks of dengue fever have occurred since 2005. A total of 4,619 dengue cases were reported between 2006 and 2020, with the highest number occurring in 2019 (1,654 cases), followed by 876 cases in 2020 [17, 22]. Recent studies have demonstrated that three dengue virus serotypes DENV-1, DENV-2 and DENV-3 are circulating in the region, with DENV-2 the most common serotype [17, 34, 35].
This study reports on the detection and sequence analysis of DENV-2 isolated from serum samples during the 2019 outbreak in Jazan region. Serotype-specific RT-PCR results yielded two groups of samples: A samples comprising DENV-2 isolates and B samples comprising DENV isolates that were negative for DENV serotyping. The Lanciotti protocol is widely used for diagnosis and surveillance of dengue [36, 37], and has been used by the SCDC in Jazan for the past decade. The present study took precautions to avoid laboratory errors and invalidate results. Furthermore, the utilised assay successfully serotyped 58.8% (173 of 294) of the samples as DENV-2. Therefore, it was hypothesised that group B samples detected during the 2019 outbreak were a new variant of DENV-2.
Subsequently, sequencing was performed to test the hypothesis and investigate the two groups' phylogenetic relationship. The sequence analysis results confirmed that A samples were DENV-2 belonging to the Cosmopolitan genotype circulating in the region since 2005 [18, 19]. Interestingly, the phylogenetic analysis for B samples revealed that these isolates were different from the Cosmopolitan genotype, with a total of 32 amino acid substitutions (Table 2, Fig. 2). Therefore, these results support the hypothesis that the DENV-2 (B samples) isolated in Jazan region during the 2019 outbreak was a new genetic variant of DENV-2 that reported for the first time in the region.
It has been suggested that genetic variations in the viral genome of fast-evolving RNA virus populations such as DENV increase the likelihood of introducing mismatches into primer or probe binding regions and result in RT-PCR false-negative results [36]. Indeed, DENV variants that escape identification with commercially available type-specific monoclonal antibodies and RT-PCR using DENV type-specific primers have been reported in other countries [23, 37–39]. However, based on sequencing and bioinformatics analyses, this was not the case in this study.
The results showed that the sequence of the newly isolated DENV-2 variant was similar to the DENV-2 isolated in some Southeast Asian countries, including Malaysia, Singapore, the Republic of Korea and China. Hence, it can be postulated that workers might import this variant from those countries when entering the region. Conversely, a new DENV-2 variant could help explain the dramatic increase in cases in 2019 and 2020, coinciding with the absence of any observed change in other factors associated with dengue transmission.
There is a scarcity of information about introduced DENV serotypes and genotypes in Saudi Arabia. Zaki et al. [18] showed that the Cosmopolitan genotype was the predominant DENV-2 genotype circulating during outbreaks in Jeddah between 1994 and 2006, and it was closely related to isolates from Australia, Thailand and Singapore. El-Kafrawy et al. [40] demonstrated that a different variant of DENV-2 Cosmopolitan genotype, with three to nine amino acid substitutions compared to other previously reported DENV-2 strains, was responsible for the outbreak in Jeddah in 2014. Thus, the high number of new variant DENV-2 isolates reported by this study is a major concern and requires continuous monitoring throughout the region.
Although the effect of genetic diversity between DENV serotypes and genotypes on clinical outcome is still controversial, the severity and endemicity of DENV have been linked to new variants in outbreaks in endemic areas [24, 41–44]. DENV-2 is the serotype commonly responsible for large epidemics in Asia and Latin America, with secondary DENV-2 infection associated with more severe disease [45–47]. The introduction of a new genetic variant was related to an increase in severe DENV cases in endemic areas [12, 48]. Unfortunately, determining the clinical severity associated with different genotypes of DENV-2 was not possible in the present study due to a lack of data. However, it is essential to monitor the spread of DENV serotypes, genotypes, and variants circulating in endemic areas to better understand the pathogenesis of DENV. Furthermore, the present study focused on the C-prM region sequence analysis. Indeed, previous studies have focused on the sequence analyses of viral individual genes or a subset of those genes, with the envelope (E) gene and C-prM region are the most frequently selected targets for DENV phylogenetic investigations and genotyping [36, 38, 49, 50]. However, a recent comparative analysis of viral mutations and evolution demonstrated significant differences when DENV whole-genome sequences were analyzed instead of individual genes [51]. Therefore, additional studies with whole DENV genome sequencing might provide critical information about the genetic relation between DENV isolates circulating in Jazan region and variants circulating in other regions.