Comparison of nucleotide sequences of colinearized fragments of structural (720 nt) and NS5 (620 nt) genes of 48 KFDV isolates from India collected over the past 5 decades (Table 1) showed a low level of diversity among these viruses (GenBank accession nos. EU29242–EU29337). A maximum of 1.2% nt and 0.5% aa differences were seen among these viruses; the most divergent virus was the A106 virus isolated in 2006. Most viruses were isolated during 1957–1972. That the 2006 virus isolate is the most divergent is consistent with the 34-year gap in sampling. As expected, little diversity was seen among the virus isolates irrespective of the host, which included humans, black-faced langurs, red-faced bonnet monkeys, various tick species (H. spinigera, H. kyasanurensis, H. turturis, H. papuana kinneari, H. wellingtoni, H. formosensis, and Ixodes petauristae), and rodents (Rattus rattus wroghtoni and R. blanfordi) (Table 1). The sequence of the 1957 KFDV reference strain (P9605) from India and strain 651521 isolated from an NIV laboratory staff member in 1965 were identical, despite their 8-year separation. However, the staff member was accidently infected while handling reference KFDV, which provided an explanation for this anomaly.

All KFDV isolates from India differed from the Alkhurma variant of KFDV (27) found in Saudi Arabia by ≈8%–9% at the nucleotide level. This finding is similar to the extent of diversity (8%) reported in a comparison of the complete genome of a KFDV isolate from India with that of an isolate from Saudi Arabia (28). In contrast, the 1989 KFDV isolate (Nanjianyin) reportedly from China (12), differed by only 1 nt (1/1,320 [0.08%]) from the 1957 KFDV reference strain (P9605) from India and the laboratory infection strain 651521. It is notable that of the 48 KFDV strains from India analyzed, the KFDV strain from China should be most similar to strain P9605, a reference strain that was distributed worldwide to arbovirus reference laboratories. The KFDV 1989 isolate from China is virtually identical at the nucleotide level to the 1957 reference strain (P9605) from India, despite their being isolated 32 years and almost 3,000 km apart, which suggests that the strain from China is not an authentic virus isolate. A reference KFDV from India appears to have been used in the analysis of serum samples from Yunnan Province (15,16), which suggests a potential source of laboratory cross-contamination. In addition, the 2 sequence fragments (EU918174 for NS5 and EU918175 for the polyprotein) of the KFDV isolate reportedly from China appear to contain several sequence analysis errors; neither fragment encodes a functional protein because of creation of a stop codon and 2 frame shifts relative to KFDV reference sequences (AY323490 and EU480689).

Bayesian coalescent analysis of sequence differences among the 48 KFDV isolates from India (1957–2006), the isolates from Saudi Arabia (1995–2004) (28,29), and the reported isolate from China (1989) (12) was conducted to estimate the rate of evolution and time to the most recent common ancestor (MRCA) for these viruses (Figure 2). These viruses were estimated to be evolving at a mean rate of 6.4 × 10^–4 substitutions/site/year (95% highest probable density [HPD] 4.1–8.8 × 10^–4 substitutions/site/year). This estimate is similar to rates for other flaviviruses analyzed by using similar Bayesian coalescent methods, including a rate of 2.17 × 10^–4 substitutions/site/year obtained for 23 St. Louis encephalitis viruses collected during 1933–2001 (30) and a rate of 4.2 × 10^–4 substitutions/site/year for yellow fever virus (31). The finding of similar evolutionary rates for tick-borne and mosquito-borne flaviviruses was unexpected, given earlier assertions that evolution of tick-borne viruses was more gradual than rapidly evolving mosquito-borne viruses (19).

Analysis estimated that the mean time to the MRCA for all the KFDV isolates was only 64 years (95% HPD 51–84 years) before 2006 (the year when the most contemporary virus was isolated). The analysis estimated that these viruses shared a common ancestor as recently as ≈1942. Analysis of only KFDV isolates from India provides a slightly more recent estimate of their MRCA (≈1948), just 9 years before identification of the disease in Kyasanur Forest in 1957. This finding correlates well with the perception of local villagers and healthcare providers in the Kyasanur Forest area that this was a newly emerged disease (32). Massive deaths of monkeys or compatible human disease in the region were not reported before the 1957 disease outbreak. In the initial years, disease activity was reported in a limited area of ≈100 km² in Sagar and Sorab taluks of Shimoga District. However, after 1972, epizootics and epidemics were recognized in several new foci, increasingly more distant from the original focus.

Most viruses analyzed were isolated in various small hamlets from migrating persons within the early enzootic zone in the Shimoga District (until 1972). Attempts to examine the relationship between genetic differences in a virus isolate relative to geographic location did not show any notable findings because of small differences and distances involved. However, the 1972 virus 72166 was isolated from a tick in the village of Gadgeri in Sirsi (Uttara Kannada District), which is north of Shimoga District. The 2006 virus A106 was isolated from a person south of Shimoga District, in Mangalore (Dakshina Kannada District) and further from the original virus epicenter (Figure 1). Although much of the topology of the virus phylogenetic tree generated by Bayesian coalescent analysis is poorly supported (nodal support posterior probability values <95), there is support for a branch that contains the 72166 1972 and the A106 2006 virus isolates (Figure 2). These data suggest that there may be an association between virus genetic divergence and temporal and geographic spread of KFDV in Karnataka, consistent with the concept of virus spreading over time from an initial focus of activity. Why this initial focus of virus activity occurred in this location and at this time remains unclear, but speculation includes emergence of the virus from a cryptic forest cycle caused by changes in land use or introduction of the virus from elsewhere by birds. A more complete picture should emerge with analysis of additional virus samples (particularly from the post-1973 period) and complete virus genomes.

Bayesian analysis estimates that the 1995 and 2004 KFDV isolates from Saudi Arabia shared a common ancestor in 1992. The node connecting these viruses with the 2006 KFDV isolate from India was in 1977, and a strongly supported node (1.0) shows that the 1972 and 2006 KFDV isolates from India shared a common ancestor with the viruses from Saudi Arabia in ≈1969. The simplest interpretation of these data and the epidemiologic observations would be that KFDV was introduced from India into Saudi Arabia in the late 1970s or the 1980s.

Similar findings of low genetic diversity and recent common ancestry were reported for the KFDVs from Saudi Arabia in a more limited study of 11 virus isolates collected over a 5-year period (1994–1999). Only 0.4%, 0.6%, and 0.9% genetic diversity were found in the E, NS3, and NS5 gene fragments of these isolates, respectively (27). Using these gene fragments along with those of the complete genome sequence of KFDV from India, the authors estimated divergence time by using an older method based on distance analysis of nonsynonymous substitutions. This estimate indicated recent ancestry of these viruses. The KFDV strains from Saudi Arabia were estimated to have diverged from one another over a 4–72 year period and the KFDVs from India and Saudi Arabia were estimated to have diverged 66–177 years ago (27).

It is unclear what factors influenced the apparent emergence of KFDV in Shimoga District, India, in 1957 and in the Makkah/Jeddah region in Saudi Arabia in 1994. Also unknown is how this tick-borne virus moved over the large distance between these regions. A considerable amount of knowledge has been accumulated with regard to the ecology of KFDV in India (32). The natural history of the virus is complex and involves dynamic cycles of various life stages of Ixodid ticks (primarily H. spinigera, but also other Haemaphysalis spp. ticks and Ixodes ticks) and amplifying (vertebrate) hosts, including rodents and shrews, and possibly monkeys and cattle. Increased human populations in the Sagar and Sorab taluks in the early 1950s may have been the primary catalyst for emergence of KFD in 1957. During 1951–1961, the population of Sagar Taluk increased 116%, bringing with it increases in deforestation, cattle grazing, and extension of paddy fields and cleared grazing areas deeper into previously forested areas (32). Expansion of the cattle population may have been a crucial factor because cattle harbor adult forms of H. spinigera ticks, and an association between cattle and increases in tick larval density has been described (32). Cattle also carry all life stages of other Haemaphysalis spp. ticks, which have been shown to be infected with KFDV. Thus, cattle would likely increase tick densities in cleared forest areas most frequented by humans. In addition, rats, shrews, and mice are highly susceptible to KFDV infection, and numerous virus isolates have been obtained from organs of infected animals (33). Changes in land use and population densities may have resulted in emergence of KFDV from a cryptic enzootic cycle in this previously heavily forested area.

A high percentage of birds in the affected area are positive for antibodies reactive with KFDV and infested with Haemaphysalis spp. and other tick genera, particularly larvae and nymphs (32). It is unclear whether birds play a role in the complex virus maintenance cycle in an enzootic zone, but birds carrying virus-infected ticks or migration of viremic birds could spread KFDV over large distances such as those separating areas of KFDV activity in India and Saudi Arabia (19,34). There is serologic evidence of KFDV, or a related flavivirus in the mammalian tick-borne virus group, in Saurashtra, Gujarat State, on the coast of India on the Arabian Sea and in birds captured outside Karnataka State (2,10,32).

The current known distribution of KFDV is limited to relatively restricted areas of India and Saudi Arabia. However, it is likely that the virus exists in other areas in cryptic enzootic cycles or is associated with unrecognized or undiagnosed disease. This finding, together with the distance separating the KFDV-affected areas in India and Saudi Arabia, despite their relatively recent common ancestry, suggests that KFD has the potential to flare up in other regions because of virus movement or ecologic changes in the area. Clinicians should consider KFD in a differential diagnosis when considering acute febrile cases with compatible symptoms in other regions of Asia and the Middle East.