The emergence of AHFV in the mid-1990s and its remarkable genetic similarity to KFDV suggested that AHFV arose from a recent introduction of KFDV from India into Saudi Arabia. Using partial E and NS5 sequences of KFDV, the AHFV/KFDV divergence was estimated to have occurred 68 years ago 
. Similarly, Charrel et al., found a TMRCA of 182 years ago with partial E, NS3, and NS5 sequences of AHFV 
. Using only the concatenated E and NS5 sequences of our isolates (as in 
), we also found a recent time of divergence (~100 years before 2009). However, analyzing full-length genome sequences, we show 2 distinct lineages, with KFDV and AHFV diverging approximately 687 years ago. Analyzing full-length genomes allowed us to incorporate the full diversity of AHFV isolates, and therefore to provide a more accurate estimate of an older common TMRCA.
The use of full genomes also revealed a slower rate of molecular evolution (9.2×10−5
substitutions/site/year) than determined previously with partial sequences of KFDV 
. This may be explained by the slightly higher NS5 and E diversity in KFDV relative to the rest of the genome (), however, the limited number of KFDV isolates available for this study might not be fully representative of the extant KFDV diversity. In accordance with the evolutionary rate patterns for vector-borne flaviviruses 
, the AHFV/KFDV rate is slower than mosquito-borne flaviviruses, including Dengue virus (DENV) 
and yellow fever virus (YFV) 
and more similar to tick-borne flaviviruses, including the closely related tick-borne encephalitis virus (TBEV Siberian subtype, 1.64×10−4
. The evolutionary rate is also similar to that of an unrelated tick-borne member of the Bunyaviridae
family, Crimean-Congo hemorrhagic fever virus (0.58–1.52×10−4
In our analyses, the sublineages of AHFV isolates did not show clustering by geography or date of isolation. Although these isolates primarily came from two distinct geographic areas, the Jeddah/Makkah region in the west and the Jizan/Najran region in the south, frequent travel of people and livestock between these regions effectively eliminates geographic boundaries. The short sequences available from the 2010 Egyptian isolate placed it within Sublineage II, the most inclusive of AHFV sublineages, possibly suggesting a relatively recent introduction to Egypt from Saudi Arabia. Alternatively, the limited sequence data available from the Egyptian isolate is not representative of extant diversity within East Africa and the full-length sequence could indicate a different evolutionary history. The narrow range of time encompassed by the viral isolates (~15 years) and the slow evolutionary rate of AHFV may also explain the lack of detectable temporal clustering.
The centuries between the divergence of the ancestral AHFV and KFDV (~year 1322), and the individual TMRCAs of AHFV (~1925) and KFDV (~1933) isolates, coupled with a slow evolutionary rate, indicate the 2 viruses evolved separately and AHFV is not a result of recent KFDV introduction in Saudi Arabia 
. This separate evolutionary history spanning several hundred years and the vast geographic distance involved raises the possibility of an existing spectrum of thus far unknown tick-borne encephalitic/hemorrhagic viruses in the regions between Saudi Arabia and India. In support of such a viral spectrum in that region, Gould et al. 
cite the presence of close AHFV/KFDV relatives, Karshi virus (KSIV) and Farm Royal virus (FRV), in Uzbekistan and Afghanistan, respectively. A similar dispersal corridor of TBEV has been described from Eastern Europe westwards 
, although more recent studies did not support such a pattern 
. Dispersal of the ancestral viruses of AHFV and KFDV may have been accomplished through the movement of animals, including camels presumably carrying ticks, along the Silk Road, which by the 1300s stretched from Europe to China.
The presence of potentially competent tick vectors in these regions 
also supports this possibility of AHFV/KFDV-like viruses existing between Saudi Arabia and India. Although the viruses are primarily associated with two tick genera, AHFV with Ornithodoros
and KFDV with Haemaphysalis
, both have been isolated from multiple genera. At least 16 tick species have been shown to transmit KFDV 
. The experimental finding of transovarial and transtadial transmission of KFDV in O. cruzi 
, coupled with the ubiquitous nature of Ornithodoros spp.
in India, Saudi Arabia and the intermediate regions, suggests these ticks may have played an important role in the spread of AHFV/KFDV-like viruses. Although the transmission of KFDV has been primarily associated with H. spinigera,
a tick species not documented in Saudi Arabia, these ticks are present between Saudi Arabia and India 
. Given the diversity of potential vectors and their extensive ranges, it is feasible that AHFV and KFDV, as well as other similar but thus far undiscovered viruses, are circulating more broadly throughout Saudi Arabia, India and beyond.
The genomic diversity of AHFV was most apparent within isolates from the tick population. Ticks sampled on the same day in one city (Najran) harbor a more genetically diverse AHFV population than isolates obtained from human cases over the course of 15 years throughout Saudi Arabia. Similarly, an analysis of the European subtype of tick-borne encephalitis virus (TBEV) isolated from ticks shows relatively high variation in a limited geographic area and temporal period, perhaps due to importation of TBEV on migratory birds 
, [ 32]
. The notable genetic heterogeneity of AHFV in Najran may be explained similarly, as camel and other livestock traders often travel large distances to Najran to market their animals. Like all soft ticks, Ornithodoros
spp. feed for only short periods, spending most of their time in burrows or nests, allowing areas such as a camel market to become a potential focus of AHFV infection and high virus genomic diversity. Presumably, like KFDV, AHFV can persist for long periods within a tick, and given the nature of RNA viruses, variants may evolve over time within the invertebrate hosts. The lower diversity within human cases may reflect limited transmissibility or reduced pathogenicity of some of these variants in people.
In our analysis, the use of full-length AHFV and KFDV genomes demonstrated a deeper evolutionary history than suggested by previous partial genome analyses. The divergence of AHFV and KFDV almost 700 years ago indicates a long period of divergent evolution, and suggests that a range of as-yet undiscovered tick-borne hemorrhagic/encephalitic viruses could exist between Saudi Arabia and India. The notably high AHFV diversity found within tick populations, coupled with the extensive geographic range of competent tick vectors, raises the possibility of broader AHFV and KFDV geographic ranges, and is supported by the recent discovery of AHFV in Egypt. As AHFV and KFDV are both associated with significant human morbidity and mortality, the potential spread of these viruses should be of serious concern and warrants further study of these significant pathogens.