**9. Phylogenesis**

Three lineages of CHIKV, with distinct genotypic and antigenic characteristics, have been identified. Isolates that caused the 2004-06 Indian Ocean outbreak form a distinct cluster within the large eastern and central Africa phylogenetic group, in addition to the Asian and west African phylogenetic groups (Powers et al., 2000; Schuffenecker et al., 2006). Phylogenetic analysis of CHIKV strains circulating in *A. Albopicus*-humans transmission cycles, obtained during outbreaks, have identified the independent acquisition of a common mutation in E1 glycoprotein (E1gp), namely A226V, in strains isolated from different geographic regions (Schuffenecker et al*.*, 2006; de Lambellerie et al., 2008). This mutation, together with M269V, D284E mutations of E1 CHIKV glycoprotein have been described as molecular signatures of the Indian Ocean outbreak (Arankalle et al., 2007; Tsetsarkin et al., 2007; Vazeille at al., 2007). In particular, the A226V mutation, which was absent in the strains isolated during the initial phases of the outbreak in Réunion, appeared in >90% of the isolates after Dicember 2005. This change could be related to virus adaptation to the mosquito vector species. Together with the lack of herd immunity, this might explain the abrupt and escalating nature of the Reunion outbreak. Has been clearly demonstrated that the A226V mutation is able to increase viral fitness in the *Aedes albopictus* vector (Tsetsarkin

Fig. 1. Geographical distribution of CHIKV shown in the most recent map coming from the

Three lineages of CHIKV, with distinct genotypic and antigenic characteristics, have been identified. Isolates that caused the 2004-06 Indian Ocean outbreak form a distinct cluster within the large eastern and central Africa phylogenetic group, in addition to the Asian and west African phylogenetic groups (Powers et al., 2000; Schuffenecker et al., 2006). Phylogenetic analysis of CHIKV strains circulating in *A. Albopicus*-humans transmission cycles, obtained during outbreaks, have identified the independent acquisition of a common mutation in E1 glycoprotein (E1gp), namely A226V, in strains isolated from different geographic regions (Schuffenecker et al*.*, 2006; de Lambellerie et al., 2008). This mutation, together with M269V, D284E mutations of E1 CHIKV glycoprotein have been described as molecular signatures of the Indian Ocean outbreak (Arankalle et al., 2007; Tsetsarkin et al., 2007; Vazeille at al., 2007). In particular, the A226V mutation, which was absent in the strains isolated during the initial phases of the outbreak in Réunion, appeared in >90% of the isolates after Dicember 2005. This change could be related to virus adaptation to the mosquito vector species. Together with the lack of herd immunity, this might explain the abrupt and escalating nature of the Reunion outbreak. Has been clearly demonstrated that the A226V mutation is able to increase viral fitness in the *Aedes albopictus* vector (Tsetsarkin

CDC's Traveler's Health website (http://wwwn.cdc.gov/travel/default.aspx).

**9. Phylogenesis** 

et al., 2007; Vazeille et al., 2007), that, in turn, may expand the potential for CHIKV to diffuse to the Americas and Europe, due to the widespread distribution of this vector, in particular in Italy (Knudsen, 1995). In a previous paper we characterized 7 viral isolates (5 imported and 2 autochthonous cases), with respect to the molecular signatures of the Indian Ocean Outbreak in E1, particularly the A226V mutation. Imported cases included 3 returning from Mauritius in 2006 and 2 returning from India in 2006 and 2007, respectively; the autochthonous cases occurred during the 2007 Italian outbreak (Bordi et al., 2008). CHIKV sequences of a 1013 bp fragment of E1 gene (nucleotide positions 10145-11158, respect to the reference strain S27) have been analyzed (Fig.2).

All 7 isolates carried the M269V and D284E Indian Ocean signatures while the A226V mutation was present in all the isolates imported from Mauritius, in the autochthonous cases from the Italian outbreak and in the isolate imported from India in 2007, but was absent in the case imported from India in 2006.

Our findings indicated that, during 2006 and 2007, multiple strains have been imported to Italy from countries where explosive Chikungunya outbreaks were ongoing. All the strains isolated in Italy, both imported and autochthonous, displayed two molecular signatures of

Fig. 2. Phylogenetic tree of CHIKV strains performed on partial E1 gene CHIKV sequences of a 1013 bp fragment of E1 gene (nucleotide positions 10145-11158, respect to the reference strain S27) have been analyzed. The strains isolated from human cases in Italy are in bold (Bordi et al., Clin Infect Dis*,* 2008)

The Re-Emergence of an Old Disease: Chikungunya Fever 129

interactions with monocytes, and with other blood leukocytes, induced a robust and rapid innate immune response with the production of specific chemokines and cytokines. In particular, high levels of IFN-were rapidly produced after CHIKV incubation with monocytes. The identification of monocytes during the early phase of CHIKV infection *in vivo* is significant as infected monocyte/macrophage cells have been detected in the synovial tissues of chronically CHIKV-infected patients, and these cells may behave as the vehicles for virus dissemination. This may explain the persistence of joint symptoms despite the

Since the A226V mutation has been associated with enhanced replication and fitness of CHIKV in *A. albopictus* vector and has also been shown to modulate cholesterol requirement for infection of insect cells (Tsetsarkin et al*.*, 2007), in a recent paper we investigated the possible involvement of A226V mutation in enhancing human pathogenesis in non vector hosts, by testing the replication competence in primate cell cultures of two isolates, differing for the presence or absence of this mutation (Bordi et al., 2011). We observed that the presence of A226V mutation did not influence the replication kinetics on primate cells. Moreover, the time course of appearance of cytopathic effect (CPE) and of cells immunostained with CHIKV-specific antiserum, was very similar for both the isolates, as well as the shape of the virus-positive multicellular foci, thus suggesting a similar

In addition, we considered the possibility that the A226V mutation could be associated with partial resistance to the inhibitory action of IFN- in classical experiments of inhibition of virus replication. Surprisingly, the A226V-carrying strain was more susceptible to the

**0.16 0 A226V IFN-α**

short duration of viraemia (Her et al., 2010).

antiviral action of recombinant IFN-. (Fig.3)

+

+ +

+

+

+

+




+

+

+

**CHIKV** 

+

+ +




mechanism of spread of the virus in the infected cell cultures.

**10,000** 

**226wt IFN α IU/ml** 

Fig. 3. Dose-dependent reduction of viral CPE by recombinant IFN-.

and the wt (Bordi et al., New Microbiol, 2011).

*In vitro* experiments of inhibition of virus replication by recombinant IFN- on Vero E6 cells showing a dose-dependent reduction of CPE for both isolates: the A226V, carrying isolate

the Indian Ocean outbreak (M269V and D284E). Concerning the A226V mutation, this was present in all imported and autochthonous cases, with the exception of the isolate imported from the Indian subcontinent in 2006. The absence of this mutation in the isolate imported in 2006 from India was in agreement with published data (Arankalle et al., 2007), and with available GenBank sequence data, indicating that the virus strains circulating in India in 2006 lacked this mutation.

The presence of A226V in the isolate imported from India in July 2007 and in the isolates from the 2007 Italian outbreak (originating from a case imported from India) supports the view that the virus envelope sequence of strains from India changed over time, acquiring after 2006 the E1 mutation associated with enhanced fitness in *Aedes albopictus*. So it appears that the acquisition and fixation of the A226V mutation may be a common pathway of chikungunya explosion in epidemic areas, in a parallel interplay with the mosquito vector dynamics. Noteworthy, the outbreak in Singapore, where the A226V mutation was absent, has been rapidly controlled.
