Hepatitis C virus (HCV) is a single-stranded, positive polarity RNA virus belonging to the genus Hepacivirus, a member of the Flaviviridae family (Li and Lo, 2015). Despite new, highly efficient antiviral drugs, HCV infection is still a major global public health problem, being a major cause of chronic hepatitis, chronic liver diseases, cirrhosis, and hepatocellular carcinoma (Bertino et al., 2016). The Middle East and Eastern Mediterranean (M and E) region have been reported to have the highest rates of HCV infection in the world, with an incidence of 62.5 per 100,000 person-years and a prevalence of 2.3% among the general population. In 2015, it was estimated that approximately one-fourth of 1.75 million newly HCV- infected persons and one-fifth of 71 million chronically infected individuals in the world resided in M and E countries. The median of the anti-HCV seropositivity rate in this region ranged broadly from 0.3% in Iran to 13.0% in Egypt. In Saudi Arabia, the seroprevalence was estimated between 0.4 and 7.3% of the general population (Abdel-Moneim et al., 2012; Alghamdi et al., 2016).

Based on genetic differences, HCV strains are classified into 8 genotypes (Borgia et al., 2018) and 93 subtypes as of March 2022 (International Committee on Taxonimy of Viruses (ICTV), 2018), which vary in geographical distribution. Studying this aspect is vital to define better HCV epidemiology, evolution, and clinically relevant therapeutic approaches. Recently, we characterized several HCV isolates from Saudi Arabia as belonging to different genotype 4 subtypes: “a” (n = 9), “o” (n = 2), “d” (n = 3), two possible recombinants (consisting of GT 4a/GT 4o/GT4a) and one patient with an unclassifiable GT 4 subtype (Di Stefano et al., 2021).

Here, we report a candidate novel HCV genotype 4 variant identified in one of these cases that showed significant genetic diversity and evolution from other characterized HCV genotype 4 isolates. The full-length genome of this novel HCV strain was obtained by using a sequence agnostic whole genome sequence approach, demonstrating that genetically divergent and unknown HCV subtypes are circulating in the human population in Saudi Arabia.

As part of a survey of HCV infection in Saudi Arabia, we previously found an unclassifiable subtype within genotype 4 in an isolate based on the sequencing of NS3, NS5A, and NS5B regions (Di Stefano et al., 2021). In agreement with the previous analysis of the three shorter regions, the complete genome sequence of the 141-HCV isolate did not cluster inside any previously known subtype, nor any reference sequences in the ICTV set that may represent other new subtypes, e.g., JX227964 and JX227963 (Figure 1A). The 141-HCV isolate formed a distinct branch with no reference sequences nearby. Furthermore, the 141-HCV isolate was at a typical genotype 4 inter-subtype distance (Figures 1B, p= 0.263, Wilcoxon rank sum test with continuity correction, using 6,483 sequences from 17 genotype 4 subtypes). We investigated the branching index (Wilbe et al., 2003) across the genome to assess the potential recombinant origin. Figure 2 shows that no part of the 141-HCV isolate genome had an origin close enough to any known subtype of genotype 4. All parts of the 141-genome sequence were far below the HCV BI threshold of 0.69, indicating a recombinant origin; thus, this isolate represents a new candidate subtype of HCV genotype 4.

HCV has been shown to have high genetic diversity and is currently classified into 8 genotypes with distinct geographical distributions. The HCV GT 4 is the most dominant in the Middle East and Central and East Africa. In contrast, HCV GT1 is the most common in North and South America, Europe, and Australia, while HCV GT 3 is the most prevalent in the Indian subcontinent. On the other hand, GT 2 is most common in West Africa, GT 5 in South Africa, and GT 6 in Southeast Asia. Finally, the newly discovered genotypes 7 and 8 are dominant in Central Africa and the subcontinent of India, respectively.

Interestingly, increased human migration leads to gradual changes in the distribution of HCV genotype, although the picture of circulating HCV diverging strains is not yet complete. Divergent HCV GT1 has been reported in Germany (Wang et al., 2019) and Cyprus (Oikonomopoulou et al., 2017), while in Canada, several divergent HCV GT 2 were observed, mainly in patients of African origin (Li et al., 2012). However, to classify it as a new HCV subtype, the characterization of three HCV isolates that cluster but are not epidemiologically linked is required (Smith et al., 2014). In the case of HCV genotype 4, four different sub-genotypes (4d, 4r, 4 L, and 4v) were reported in Ethiopia (Hundie et al., 2017). Although subgenotype 5a is the most predominant in South Africa, other subgenotypes and recombinant viruses could be found (Simmonds et al., 2005), and these recombination events could play a significant role in the evolution of RNA viruses. We should also consider that HCV GT 4 epidemiology is changing. It started to expand beyond its strongholds in Africa and the Middle East to several Western countries, especially in Europe, due to changes in demographic structure, immigration, and injection drug use (de Bruijne et al., 2009; Ciccozzi et al., 2012).

Understanding the different genotypes and subtypes of HCV is crucial as they can significantly impact the success of treatment and clinical outcomes of HCV infections (Schinazi et al., 2014; Waldenström et al., 2016). For instance, Interferon-based therapy has shown higher success rates for GT 2 and 3 (Waldenström et al., 2016), while the first- generation HCV protease inhibitors were more effective in GT 1 (Schinazi et al., 2014). Fortunately, second-generation direct acting antivirals have broader coverage across genotypes. However, despite the promise of pangenotypic regimens, they are expensive and often unavailable in low or middle-income countries. As a result, access to these treatments becomes a challenge for many patients in these regions.

These findings demonstrate that through phylogenetic reconstructions using the entire genome of HCV-4 strains, we found that the 141-HCV isolate formed a distinct cluster within the HCV-4 group. This provides valuable new information about the variability of HCV and sheds light on the unique characteristics of this particular strain.

The data presented in the study are deposited in the GenBank repository, accession number OR594285.

This study was performed according to the Declaration of Helsinki, and ethical approval was obtained from Imam Abdulrahman Bin Faisal University (IRB-2020-01-313, 26 October 2020). The study was conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

MD: Conceptualization, Supervision, Writing – original draft, Writing – review & editing. MI: Data curation, Investigation, Writing – review & editing. TL: Formal analysis, Funding acquisition, Investigation, Writing – review & editing. GF: Methodology, Investigation, Writing – original draft. JLM: Formal analysis, Investigation, Writing – review & editing. MT: Investigation, Methodology, Writing – review & editing. JF: Conceptualization, Supervision, Writing – original draft, Writing – review & editing. TS: Writing – original draft, Writing – review & editing.