**6. Treatment**

Currently, there are no available specific therapeutics against CHIKV. Treatment is purely symptomatic and can include rest, fluids, and medicines to relieve symptoms of fever and aching, such as ibuprofen, naproxen, acetaminophen, or paracetamol. Non-steroidal antiinflammatory drugs (NSAIDs) are primarily used to treat inflammation but high doses, administrated to control arthralgia, could cause thrombocytopenia, gastrointestinal bleeding, nausea, vomiting and gastritis (Jain et al, 2008; Pialoux et al., 2007). Steroids have been occasionally used but their efficacy was not significant (Taubitz et al., 2007). Some time ago chloroquine, a drug useful for prophylaxis and treatment of malaria, showed promising results for treating chronic Chikungunya arthritis (Brighton, 1984), while a recent trial conducted on French Reunion Island proved that there is currently no justification for the use of chloroquine to treat acute chikungunya diseases (De Lamballerie et al., 2008). However, the usefulness of chloroquine in the treatment of Chikungunya infection deserves further investigation that could take advantage on the availability of a non-human primate animal model (Labadie et al., 2010). Ribavirin (200 mg twice a day for seven days) given to

The Re-Emergence of an Old Disease: Chikungunya Fever 123

mutations, alternative genetic strategies such as viral chimeras offer the promise of more stable attenuation (Kennedy et al., 2011). In addition to the risk of reactogenicity, attenuation based on small numbers of mutations can also result in residual alphavirus infectivity for mosquito vectors. This risk, which was underscored by the isolation of the TC-83 VEEV vaccine strain from mosquitoes in Louisiana during an equine vaccination campaign designed to control the 1971 epidemic (Pedersen et al., 1972), is especially high when a vaccine that relies on a small number of point mutations is used in a nonendemic location that could support a local transmission cycle. In a recent study chimeric alphaviruses, encoded CHIKV-specific structural genes (but no structural or nonstructural proteins capable of interfering with development of cellular antiviral response) induced protective immune response against subsequent CHIKV challenge (Wang et al., 2011). More in detail, recombinant chikungunya virus vaccine, comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus, consistently induced in mice high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic (Wang et al., 2011). A novel CHIK vaccine candidate, CHIKV/IRES, was generated by manipulation of the structural protein expression of a wt-CHIKV strain via the EMCV IRES (Plante et al., 2011). In particular, the internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. This vaccine candidate exhibited a high degree of murine attenuation that was not dependent on an intact

interferon type I response, highly attenuated and efficacious after a single dose.

humoral immune responses in either case.

vaccine to prevent this disease will not be too long in arriving.

Another approach was the selective expression of CHIK viral structural proteins recently obtained by Akata and collegues using virus-like particles (VLPs) *in vitro,* that resemble replication-competent alphaviruses (Akahata et al., 2010). Immunization of monkeys with these VLPs elicited neutralizing antibodies against envelope proteins from different CHIKV strains and obtained antibodies transferred into mice protective against subsequent lethal CHIKV challenge. The last frontier in the approach of CHIK vaccine design is the DNA vaccine strategy. An adaptive constant-current electroporation technique was used to immunize mice (Muthumani et al., 2008) and rhesus macaques (Mallilankaraman et al., 2011) with an intramuscular injection of plasmid coding for the CHIK-Capsid, E1 and E2. Vaccination induced robust antigen-specific cellular and

To date a number of CHIKV vaccines have been developed, but none have been licensed. While a number of significant questions remain to be addressed related to vaccine validation, such as the most appropriate animal models (species, age, immune status), the dose and route of immunization, the potential interference from multiple vaccinations against different viruses, and last, the practical cost of the vaccine, since most of the epidemic geographical regions belong to the developing countries, there is real hope that a

Since a vaccine is not available actually, protection against mosquito bites and vector control are the main preventive measures. Individual protection relies on the use of mosquito repellents and measures in order to limit skin exposure to mosquitoes. Bednets should be used during the night in hospitals and day-care facilities but *Aedes* mosquitoes are active allday-long. Control of both adult and larval mosquito populations uses the same model as for dengue and has been relatively effective in many countries and settings. Breeding sites must be removed, destroyed, frequently emptied, and cleaned or treated with insecticides. Large-

patients who continued to have crippling lower limb pains and arthritis for at least two weeks after a febrile episode, had a direct antiviral property against CHIKV, leading to faster resolution of joint and soft tissue manifestations (Ravichandran & Manian, 2008). Briolant and collegues screened various active antiviral compounds against viruses of the Alphavirus genus *in vitro* and demonstrated that 6-azauridinet was more effective against CHIKV, as compared to ribavirin. Moreover, the combination of IFN-alpha2b and ribavirin had synergistic antiviral effect on Chikungunya virus (Briolant et al., 2004).

It is widely recognized that passive vaccination is an appropriate preventive and therapeutic option for many viral infections in human, including those spread by viral vertical transmission, especially when no alternative therapy is available (Dessain et al., 2008). Human polyvalent immunoglobulins purified from plasma samples obtained from donors in the convalescent phase of CHIKV infection exhibited a high *in vitro* neutralizing activity and a powerful prophylactic and therapeutic efficacy against CHIKV infection *in vivo* in mouse models (Couderc et al., 2009). Due to the demonstrated efficacy of human anti-CHIKV antibodies in a mouse model, purified polyvalent CHIKIg (commercialized under the brand Tégéline) could be used in humans for prevention and treatment, especially in individuals at risk of severe CHIKV disease, such as neonates born to viraemic mothers and adults with underlying conditions. Polyclonal immune globulins present the advantage of a broad reactivity but the therapeutic intervention is limited, due to the short viremia in acute phase of CHIKV infection: thus the only benefit this treatment has to offer would be to help reducing viremia faster (Kam et al., 2009). As an alternative, more specific human monoclonal antibodies (MAbs) could be used. In a recent study two unique human mAbs, specific for the CHIKV envelope glicoproteins, strongly and specifically neutralized CHIKV infection *in vitro* (Warter et al., 2011).

#### **7. Prevention**

Although no licensed vaccines are currently available for CHIKV, potential vaccine candidates have been tested in humans and animals with varying success. Due to the easiness in preparation, the first developed vaccines were formulations of whole-virus grew on cells and inactivated either by formalin or tween-ether (Eckels et al., 1970; Harrison et al., 1967, 1971; White et al., 1972).

Further vaccines are focused on attenuated strains of CHIK obtained after serial passages in cells cultures (Edelman et al., 2000; Levitt et al., 1986). One of these promising candidates is TSI-GSD-218, a serially passaged and plaque-purified live CHIK vaccine, tested for safety and immunogenicity in human Phase II trials by the US Army Medical Research Institute (Edelman et al., 2000). Seroconversion was obtained in 98% of vaccinees volunteers by day 28 and neutralizing antibodies persisted in 85% of cases at one year after immunization. However transient arthralgia occurred in 8% of the volunteers. Some chimeric candidates vaccines were developed using either Venezuelan equine encephalitis (VEEV) attenuated vaccine strain TC-83, a naturally attenuated strain of eastern equine encephalitis virus (EEEV), or Sindbis virus (SV) as a backbone and the structural protein genes of CHIKV. Vaccinated mice were fully protected against disease and viraemia after CHIKV challenge (Wang et al., 2008). The maturity of reverse genetic technology has provided unprecedented opportunities for manipulation of the alphaviral genome to improve attenuation strategies. Thus, unlike traditional attenuation approaches that rely on cell culture passages, which typically result in attenuation that depends only on small numbers of attenuating point

patients who continued to have crippling lower limb pains and arthritis for at least two weeks after a febrile episode, had a direct antiviral property against CHIKV, leading to faster resolution of joint and soft tissue manifestations (Ravichandran & Manian, 2008). Briolant and collegues screened various active antiviral compounds against viruses of the Alphavirus genus *in vitro* and demonstrated that 6-azauridinet was more effective against CHIKV, as compared to ribavirin. Moreover, the combination of IFN-alpha2b and ribavirin

It is widely recognized that passive vaccination is an appropriate preventive and therapeutic option for many viral infections in human, including those spread by viral vertical transmission, especially when no alternative therapy is available (Dessain et al., 2008). Human polyvalent immunoglobulins purified from plasma samples obtained from donors in the convalescent phase of CHIKV infection exhibited a high *in vitro* neutralizing activity and a powerful prophylactic and therapeutic efficacy against CHIKV infection *in vivo* in mouse models (Couderc et al., 2009). Due to the demonstrated efficacy of human anti-CHIKV antibodies in a mouse model, purified polyvalent CHIKIg (commercialized under the brand Tégéline) could be used in humans for prevention and treatment, especially in individuals at risk of severe CHIKV disease, such as neonates born to viraemic mothers and adults with underlying conditions. Polyclonal immune globulins present the advantage of a broad reactivity but the therapeutic intervention is limited, due to the short viremia in acute phase of CHIKV infection: thus the only benefit this treatment has to offer would be to help reducing viremia faster (Kam et al., 2009). As an alternative, more specific human monoclonal antibodies (MAbs) could be used. In a recent study two unique human mAbs, specific for the CHIKV envelope glicoproteins, strongly and specifically neutralized CHIKV

Although no licensed vaccines are currently available for CHIKV, potential vaccine candidates have been tested in humans and animals with varying success. Due to the easiness in preparation, the first developed vaccines were formulations of whole-virus grew on cells and inactivated either by formalin or tween-ether (Eckels et al., 1970; Harrison et al.,

Further vaccines are focused on attenuated strains of CHIK obtained after serial passages in cells cultures (Edelman et al., 2000; Levitt et al., 1986). One of these promising candidates is TSI-GSD-218, a serially passaged and plaque-purified live CHIK vaccine, tested for safety and immunogenicity in human Phase II trials by the US Army Medical Research Institute (Edelman et al., 2000). Seroconversion was obtained in 98% of vaccinees volunteers by day 28 and neutralizing antibodies persisted in 85% of cases at one year after immunization. However transient arthralgia occurred in 8% of the volunteers. Some chimeric candidates vaccines were developed using either Venezuelan equine encephalitis (VEEV) attenuated vaccine strain TC-83, a naturally attenuated strain of eastern equine encephalitis virus (EEEV), or Sindbis virus (SV) as a backbone and the structural protein genes of CHIKV. Vaccinated mice were fully protected against disease and viraemia after CHIKV challenge (Wang et al., 2008). The maturity of reverse genetic technology has provided unprecedented opportunities for manipulation of the alphaviral genome to improve attenuation strategies. Thus, unlike traditional attenuation approaches that rely on cell culture passages, which typically result in attenuation that depends only on small numbers of attenuating point

had synergistic antiviral effect on Chikungunya virus (Briolant et al., 2004).

infection *in vitro* (Warter et al., 2011).

1967, 1971; White et al., 1972).

**7. Prevention** 

mutations, alternative genetic strategies such as viral chimeras offer the promise of more stable attenuation (Kennedy et al., 2011). In addition to the risk of reactogenicity, attenuation based on small numbers of mutations can also result in residual alphavirus infectivity for mosquito vectors. This risk, which was underscored by the isolation of the TC-83 VEEV vaccine strain from mosquitoes in Louisiana during an equine vaccination campaign designed to control the 1971 epidemic (Pedersen et al., 1972), is especially high when a vaccine that relies on a small number of point mutations is used in a nonendemic location that could support a local transmission cycle. In a recent study chimeric alphaviruses, encoded CHIKV-specific structural genes (but no structural or nonstructural proteins capable of interfering with development of cellular antiviral response) induced protective immune response against subsequent CHIKV challenge (Wang et al., 2011). More in detail, recombinant chikungunya virus vaccine, comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus, consistently induced in mice high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic (Wang et al., 2011).

A novel CHIK vaccine candidate, CHIKV/IRES, was generated by manipulation of the structural protein expression of a wt-CHIKV strain via the EMCV IRES (Plante et al., 2011). In particular, the internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. This vaccine candidate exhibited a high degree of murine attenuation that was not dependent on an intact interferon type I response, highly attenuated and efficacious after a single dose.

Another approach was the selective expression of CHIK viral structural proteins recently obtained by Akata and collegues using virus-like particles (VLPs) *in vitro,* that resemble replication-competent alphaviruses (Akahata et al., 2010). Immunization of monkeys with these VLPs elicited neutralizing antibodies against envelope proteins from different CHIKV strains and obtained antibodies transferred into mice protective against subsequent lethal CHIKV challenge. The last frontier in the approach of CHIK vaccine design is the DNA vaccine strategy. An adaptive constant-current electroporation technique was used to immunize mice (Muthumani et al., 2008) and rhesus macaques (Mallilankaraman et al., 2011) with an intramuscular injection of plasmid coding for the CHIK-Capsid, E1 and E2. Vaccination induced robust antigen-specific cellular and humoral immune responses in either case.

To date a number of CHIKV vaccines have been developed, but none have been licensed. While a number of significant questions remain to be addressed related to vaccine validation, such as the most appropriate animal models (species, age, immune status), the dose and route of immunization, the potential interference from multiple vaccinations against different viruses, and last, the practical cost of the vaccine, since most of the epidemic geographical regions belong to the developing countries, there is real hope that a vaccine to prevent this disease will not be too long in arriving.

Since a vaccine is not available actually, protection against mosquito bites and vector control are the main preventive measures. Individual protection relies on the use of mosquito repellents and measures in order to limit skin exposure to mosquitoes. Bednets should be used during the night in hospitals and day-care facilities but *Aedes* mosquitoes are active allday-long. Control of both adult and larval mosquito populations uses the same model as for dengue and has been relatively effective in many countries and settings. Breeding sites must be removed, destroyed, frequently emptied, and cleaned or treated with insecticides. Large-

The Re-Emergence of an Old Disease: Chikungunya Fever 125

Germany, Greece, Italy, Serbia, Spain, Switzerland, Norway, and the United Kingdom (Beltrame, A. 2007; Deporteere & Coulombier, 2006; Fusco, F.M. 2006; Pialoux et al., 2007; Taubitz et al., 2007). In 2006, CHIK fever cases have also been reported in traveller returning from known outbreak areas to Canada, the Caribbean (Martinique), and South America (French Guyana). During 2005-2006, 12 cases of CHIK fever were diagnosed serologically and virologically at CDC in travellers who arrived in the United States from areas known to be epidemic or endemic for CHIK fever, and 26 additional imported cases with onset in 2006 underscores the importance of recognizing such cases among travellers

Moreover, CHIKV gave rise in 2007 to the first autochthonous European outbreak in Italy, in

In June 2007, an Indian citizen returned to Italy after a visit to relatives in Kerala, India, developed 2 episodes of fever. During the second febrile episode, he visited his cousin in Castiglione di Cervia. The cousin had an onset of symptoms, with fever and arthralgia, on July 4. This sequence of events started the first Chikungunya fever outbreak in a temperate country, that lasted approximately 2 months with a total 247 cases of Chikungunya fever occurred in the region (217 laboratory-confirmed, 30 suspected) (Fusco et al., 2010). A unique sequence of events seems to have contributed to the establishment of local transmission in Emilia-Romagna: the high concentration of competent vectors *A. albopictus*  in the area at the time of arrival of the index case, the presence of a sufficient human population density and the temporal overlapping of arthropod activity (seasonal

During 2008, cases of Chikungunya fever have been reported from many countries in Asia other than India, as well as active epidemics from Singapore, Sri Lanka, and Malaysia (Leo

Since 2006, the Regional Office of the French Institute For Public Health Surveillance in the Indian Ocean has conducted epidemiological and biological surveillance for CHIKV infection. During the period December 2006-July 2009, no confirmed case was detected on Reunion Island and Mayotte, but new outbreaks were reported in Madacascar. After few years of relative dormancy in Réunion Island, in August 2009, a cluster of cases was identified on the western coast of Réunion Island (D'Ortenzio et al., 2009) and, subsequently, an outbreak of CHIKV infection was described on Réunion Island in 2010 (D'Ortenzio et al., 2011). Moreover, recent publications described cases of Chikungunya fever in tourist returning from Maldives, confirming the circulation of the virus by the end

These episodes have refreshed the concerns about the possibility of renewed autochthonous transmission in Mediterranean countries and highlight the need for surveillance in countries where emerging infections may be introduced by returning travellers. Travellers can serve as sentinel population providing information regarding the emergence or re-emergence of an infectious pathogen in a source region. Travellers can thus act as carriers who inadvertently ferry pathogens that can be used to map the location, dynamics and movement of pathogenic strains (Pistone et al, 2009). Thus, with the increase in intercontinental travel, travellers can provide insights into the level of the risk of

the northern region of Emilia-Romagna (Rezza et al*.,* 2007; Charrel et al., 2008).

syncronicity) (Charrel et al., 2008; Rezza et al*.,* 2007).

of 2009 (Pfeffer et al., 2010; Receveur et al., 2010)

transmission of infections in other geographical regions.

The geographic range of CHIKV is mainly in Africa and Asia (Fig. 1)

(CDC, 2006; CDC 2007).

et al., 2009).

scale prevention campaigns using DDT have been effective against *A. aegypti* but not *A. albopictus*. Control of *A. aegypti* has rarely been achieved and never sustained (Reiter et al., 2006). Recent data show the different degrees of insecticide resistance in *A. albopictus* and *A. aegypti* (Cui et al., 2006). However, vector control is an endless, costly, and labour-intensive measure and is not always well accepted by local populations, whose cooperation is crucial. Control of CHIKV infection, other than use of drugs for treatment of disease, development of vaccines, individual protection from mosquitoes and vector control programs, also involves surveillance that is fundamental for early identification of cases and quarantine measurement. A model used in investigation of the transmission potential of CHIKV in Italy has proven useful to provide insight into the possible impact of future outbreaks in temperate climate regions and the effectiveness of the interventions performed during the outbreak (Poletti et al., 2011).
