2.2. Virus structure and genomic organisation

The virion has an icosahedral capsid enclosed by a lipid envelope and a diameter of 60–70 nm. It is sensitive to desiccation and to temperatures >58 °C. The genome is a single-stranded, positive sense, RNA molecule of approximately 12 kb in length (Khan et al., 2002) (Fig. 2 ). The genomic organisation is arranged as with others alphaviruses: 5′-nsP1–nsP2–nsP3–nsP4-junction region-C–E3–E2–6k–E1-poly (A)-3′ with two open reading frames (ORFs). The 5′ end of the genome has a 7-methylguanosine cap and there is a polyadenylation signal at the 3′ end. The 5′ ORF is translated from genomic RNA and encodes four non-structural proteins (nsP1, 2, 3 and 4) (Jose et al., 2009). The 3′ ORF is translated from a subgenomic 26S RNA and encodes a polyprotein that is processed as the capsid protein (C), two surface envelope glycoproteins (E1 and E2) and two small peptides designated E3 and 6k (King et al., 2012, Simizu et al., 1984, Voss et al., 2010).

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Fig. 2

Organisation of the CHIKV genome and gene products. The genomic organisation is arranged as with other alphaviruses, with two open reading frames (ORFs), 5′ cap structures and a 3′ poly(A) tail. The 5′ and 3′ proximal sequences of the CHIKV genome include nontranslated regions (NTR). The junction region (J) is also noncoding. The 5′ ORF is translated from genomic RNA and encodes four nonstructural proteins (nsP1, 2, 3 and 4). The 3′ ORF is translated from a subgenomic 26S RNA and encodes the capsid protein (C), two surface envelope glycoproteins (E1 and E2) and two small peptides designated E3 and 6k. The different non-structural proteins (nsP1–nsP4) and structural proteins (C, Capsid; E1, E2, E3, envelope; 6k) are generated after proteolytic cleavage of polyprotein precursors (adapted from Solignat et al., 2009, with authors’ permission).

The glycoproteins E1 and E2 are embedded in a heterodimeric form in the viral envelope and are responsible for virus attachment and membrane fusion. Virus fusion with the cell membrane is mediated by the E1 glycoprotein, a class II fusion protein, in a process dependent on low-pH. Acidic conditions induce a conformational change in the virus envelope proteins, dissociation of the E2–E1 heterodimers and formation of E1 homotrimers. The E1 trimer is inserted into the target membrane via its hydrophobic fusion peptide and refolds to form a hairpin-like structure. Cholesterol is required for both cell membrane fusion and budding during alphavirus infection (Solignat et al., 2009). A large number of more recent studies have been dedicated to the structural characterisation of the envelope proteins of alphaviruses and to structural modifications that occur during fusion (Gibbons et al., 2004, Li et al., 2010, Liu and Kielian, 2009, Roussel et al., 2006, Voss et al., 2010, Zhang et al., 2011).

3.2. Mild cases and asymptomatic infections

The proportion of individuals infected by CHIKV who develop clinical symptoms requiring medical attention is higher than in most other arboviral infections (Chastel, 2011). Following outbreaks of chikungunya fever, several recent seroprevalence studies were performed indicating different rates of asymptomatic cases ranging from 3.8% to 27.7%. After the outbreak in Reunion island, 162/967 (16.7%) cases reported no symptoms including 46/967 (4.8%) who ‘did not know’ (Gerardin et al., 2008b). Several months after the outbreak in Italy, 6/33 (18.2%) cases with positive serology declared no symptoms (Moro et al., 2010). Similarly, after the outbreak in Mayotte, 122/440 (27.7%) cases with positive serology declared no clinical signs that could be compatible with a history of chikungunya fever (Sissoko et al., 2010).

Two years after the chikungunya fever outbreak in the state of Kerala in India, a seroprevalence study found that amongst the 260 patients with positive serology, only 10 (3.8%) declared no symptoms compatible with a history of chikungunya fever (Kumar et al., 2011). In a study amongst French military policemen after the chikungunya outbreak in Reunion island, only 3.2% of cases did not report symptoms (Queyriaux et al., 2008). Finally in a case-control study in Thailand, 9% of laboratory-proven CHIKV infection cases were considered as asymptomatic and the viral loads observed in the symptomatic individuals were not significantly different from those observed in the viremic asymptomatic individuals (Appassakij et al., 2012).

3.3. Clinical features of chikungunya fever

Since the Indian Ocean outbreak in 2005–2006, the information available for the scientific community relating specifically to the clinical characteristics of patients infected by CHIKV has significantly increased. Two stages of the disease are now described: acute illness and the late stage of illness, with persistent arthropathy.

3.4. Atypical cases

Since the 1960–1970s CHIKV has been known to affect the central nervous system. During the recent Indian Ocean outbreak, neurological complications were reported in less than 25% of cases. Seizures were most often reported in patients who had a past medical history of epilepsy and/or in those with a history of heavy alcohol consumption. Other neurological complications reported are encephalopathy, encephalitis, Guillain–Barre, encephalomyeloradiculitis or subarachnoid cerebellar haemorrhages (Borgherini et al., 2007, Das et al., 2010, Staikowsky et al., 2009).

Haemorrhagic signs are rare (1–7% of patients) and minor, such as bleeding of the nose and gums (Borgherini et al., 2007, Win et al., 2010). In most cases, they are not associated with clotting abnormalities or major thrombocytopenia (Nkoghe et al., 2012, Staikowsky et al., 2009).

A variety of other clinical symptoms have been reported during the acute stage of chikungunya fever, such as conjunctivitis, neuroretinitis, iridocyclitis, myocarditis, pericarditis, pneumonia, dry cough, lymphadenopathy, nephritis, hepatitis and pancreatitis (Borgherini et al., 2007, Economopoulou et al., 2009, Mahendradas et al., 2008, Mirabel et al., 2007, Nair et al., 2012, Rajapakse et al., 2010, Renault et al., 2007, Rezza et al., 2007, Rose et al., 2011, Simon et al., 2007, Simon et al., 2008, Staikowsky et al., 2009, Win et al., 2010). Overall, atypical cases were estimated at 0.3% of all symptomatic cases during the 2006 Reunion island outbreak (Economopoulou et al., 2009). Amongst these atypical cases, 36% were considered to be severe, 14% were admitted to an intensive care unit and 10% died (Economopoulou et al., 2009). The reported causes of death were heart failure, multiple organ failure syndrome, toxic hepatitis, encephalitis, bullous dermatosis, respiratory failure, renal failure, pneumonia, acute myocardial infarction, cerebrovascular disease, hypothyroidism or septicaemia.

During the Reunion island outbreak, an excessive death-rate was observed and mortality associated with chikungunya fever was reported to have had a case-fatality ratio of about 1 in 1000 (Josseran et al., 2006). A similar case-fatality ratio was observed following the outbreak in Port Blair, capital city of the Union Territory of Andaman and Nicobar Islands, India, in 2006 (Manimunda et al., 2011). However, the comparison between expected and observed mortality in Reunion island, 2006, identified a high-mortality rate between January and May and a low-mortality rate during the rest of the year (Renault et al., 2012), suggesting the existence of a ‘harvesting’ phenomenon and implying that the global excess of death might have been overestimated when derived only from the epidemic period.

3.5. Chikungunya fever in children

In children, clinical manifestations of chikungunya fever appear to be quite specific and although rheumatological manifestations are less frequent, they remain a group at high risk of atypical or severe manifestations. The main clinical characteristics in infants are the high prevalence of dermatological manifestations (hyperpigmentation, generalised erythema, maculopapular rash and vesiculobullous lesions) and neurological complications (encephalitis, seizures, meningeal syndrome or acute encephalopathy). Other clinical features are also described such as digestive disorder (loose stools), peripheral cyanosis and minor haemorrhagic manifestations (Robin et al., 2008, Robin et al., 2010, Sebastian et al., 2009, Valamparampil et al., 2009).

3.6. Chikungunya fever in pregnant women

Although chikungunya fever apparently has no observable teratogenic effects during pregnancy (Fritel et al., 2010), vertical transmission was reported for the first time, during the 2006 Reunion island outbreak and was observed exclusively in near-term deliveries in the context of intrapartum viremia, with a rate of 49%. Caesarean section had no protective effect on transmission. Severe illness was observed in 53% of newborns and mainly consisted of encephalopathy with persistent disabilities in 44% of them (Gerardin et al., 2008a). The others complications included seizures, haemorrhagic syndrome, haemodynamic disorders, cardiologic complication (myocardial hypertrophy, ventricular dysfunction, pericarditis, coronary artery dilatation), necrotizing enterocolitis or dermatologic manifestation (Nair, 2008, Ramful et al., 2007, Rao et al., 2008).

3.7. Risk factors for severe disease

Risk factors for severe acute disease have been thoroughly investigated (Borgherini et al., 2007, Chow et al., 2011, Economopoulou et al., 2009, Gerardin et al., 2008a, Gerardin et al., 2008c, Kam et al., 2012c, Kee et al., 2010, Kumar et al., 2010, Lohachanakul et al., 2012, Ng et al., 2009b, Nkoghe et al., 2012, Paquet et al., 2006, Renault et al., 2007, Sissoko et al., 2008, Staikowsky et al., 2009). Several studies reported that, in adults, the incidence of atypical cases, severe cases, hospitalisation and the mortality rate increased with age. In pediatric populations, newborns had a high risk of severe disease. Although comorbidity and increase of age are often linked, comorbidity or underlying respiratory diseases, the use of NSAIDs prior to hospitalisation, hypertension and underlying cardiac disorders have been associated with hospitalisation or disease severity. Alcohol abuse was also associated with increased mortality.

Female gender has not been clearly associated with more severe illness but, during the Indian Ocean outbreaks, women were over-represented based on reported cases, whereas cross-sectional studies found similar seroprevalence values for both genders, suggesting a variability of symptomatic disease depending on gender. In contrast, in a single study, males and blood Rhesus-positive individuals were found to be more susceptible to infection by CHIKV. Case reports of immunocompromised patients have described atypical and severe disease but no studies have compared immunocompromised or immunodefficient patients. The association between viral load and acute severe illness is controversial. Indeed, several studies reported higher viral load in hospitalised patients or patients with severe illness whilst others studies did not reveal significant association between the viral load and the clinical presentation. Other biological abnormalities or immunological markers have been proposed to be associated with disease severity such as elevated liver enzyme, creatinine, C reactive protein (CRP), hypocalcemia, elevated IL-1β or IL-6, decreases in RANTES or early CHIKV-specific IgG3.

4.2. Vertebrate reservoirs and transmission cycle

Human beings serve as reservoir hosts for the virus during epidemic periods whereas during inter-epidemic periods, several other reservoir hosts have been incriminated such as monkeys, rodents and birds (Inoue et al., 2003, Wolfe et al., 2001). The significance of non-human primates as reservoir hosts is discussed in the section devoted to pathogenesis.

Two distinct transmission cycles have been described for CHIKV: a sylvatic cycle in Africa (Wolfe et al., 2001), the general pattern of which resembles the yellow fever virus sylvatic cycle (i.e., involving virus transmission between forest-or savannah-associated mosquitoes and non-human primates (and possibly rodents) with occasional spillover of the virus into nearby human populations living in or close to the sylvatic environment). Under these latter circumstances the virus may then encounter mosquitoes primarily associated with urban environments (i.e., domestic or peridomestic mosquitoes) thus initiating an urban human–mosquito–human virus transmission cycle such as those seen in Asia, the Indian Ocean, Africa and more recently, Europe (Rezza et al., 2007). In general, when Ae. aegypti is the predominant vector, these urban outbreaks or epidemics resemble the virus transmission cycle of dengue virus in the urban environment.

In Africa, CHIKV is maintained in a sylvatic cycle involving wild non-human primates and a variety of forest-dwelling mosquito species (Powers and Logue, 2007), such as Ae. africanus in Uganda and Bangui, Ae. cordellieri in South Africa, Ae. furcifer–taylori in South Africa and Senegal (Diallo et al., 1999) and Ae. luteocephalus and Ae. dalzieli in Senegal (Diallo et al., 1999, Jupp and McIntosh, 1990, McIntosh et al., 1963). In rural regions of Africa, the outbreaks tend to affect small populations and appear to be heavily dependent on the sylvatic mosquito population densities that increase during periods of heavy rainfall (Diallo et al., 1999, Thonnon et al., 1999). However, during recent outbreaks in Cameroon, Congo and Gabon, CHIKV was vectored by the Asian mosquito Ae. albopictus which has gradually widened its geographic range, out of Asia into Africa, Europe and the Americas (Benedict et al., 2007). Currently, the principle factors (if indeed there are any) responsible for the maintenance of this Ae. albopictus-adapted virus life cycle in the sylvatic environment have not been defined.

In Asia, CHIKV is maintained in human-mosquito-human urban cycles involving both Ae. aegypti and Ae. albopictus. Whether or not these transmission cycles are exclusive for each mosquito species is not yet known and has not yet been fully investigated. For many years, CHIKV persistence was thought to depend primarily upon continuous introductions of the virus into immunologically naïve populations (Powers and Logue, 2007). However, the evidence for long lasting persistence of the Asian genotype (see infra) and several recent studies suggest that sylvatic cycles could also play a part in the transmission of CHIKV in Asia (Powers et al., 2000, Apandi et al., 2009).

7.1. Single recombinant antigens or Vero cell-adapted, formalin-inactivated vaccine

A vaccine based on recombinant envelope proteins of an Indian strain of CHIKV, adjuvanted with Alum, FCA or Montanide, elicited a balanced Th1/Th2 response with virus-neutralising antibodies in BALB/C mice, but the authors did not further challenge these mice (Khan et al., 2012). Kumar et al. (2012) evaluated recombinant E2 protein-based and whole-virus inactivated candidate vaccines, given intramuscularly, against CHIKV in BALB/c mice. They tested 4 adjuvants: Alum, Mw, CadB (rE2p), Alum/Mw (formalin-inactivated CHIKV) and Alum (BPL-inactivated CHIKV). Humoral immunity was assessed by ELISA and in vitro neutralisation test, using homologous and heterologous (Asian genotype) strains of CHIKV.

Two cohorts of vaccinated mice were challenged separately via the intranasal route with homologous virus 2 and 20 weeks after the second dose. Anti-CHIKV-antibody titres were dose dependent for all the immunogenic formulations. BPL-inactivated vaccines led to the highest ELISA/neutralising antibody (nAb) titres, whilst alum was the most effective adjuvant. In this adult BALB/c mouse/intra-nasal infection model (6–8 weeks), complete protection was observed following immunisation with the alum-adjuvanted rE2p, and both the inactivated vaccines, as no virus was detected in the tissues (muscle, brain, spleen) and blood (real time PCR) after challenge 2 weeks or 20 weeks-post-the second vaccine dose. However, with rE2p–CadB, very low viremia was recorded on the second day-post-challenge. Globally, a majority of vaccination trials in mice, even with the very simple inactivation system of whole CHIKV particles inactivated with aziridine (Brown, 2001), gave effective protection with as low as 0.01 μg of vaccine in C57BL6/adult mice (12 weeks of age) with suppression of viremia and leg oedema.

7.2. Structural components of CHIKV in non-infectious virus-like particles (VLPs)

VLPs representing CHIKV envelope protein were produced in 293A renal epithelial cells and 293T human kidney cell lines infected by lentivirus vectors containing the subgenomic RNA sequence (C–E3–E2–6k–E1) (Akahata et al., 2010). The authors showed that, using VLP plus Ribi adjuvant, an antibody response 20 times higher than when using a DNA vaccine with the same sequence in Balb/C mice could be detected. Finally they reported protection in both mice (Ifnar −/−) and non-human primates challenged with the LR-2006 OPY-1 strain of CHIKV. However, the amount of produced VLPs remained very low in this system: from 0.1 to 20 mg/l within the 293T supernatant medium. The method was improved recently when the authors showed that a specific domain of the CHIKV E2 protein (amino-acid 233N–234K–252Q) regulated particle formation in human cells (Akahata and Nabel, 2012) and thus production of functional VLPs might be enhanced up to 200 fold.

A group from Wageningen University developed production of VLPs using chimeric baculovirus with the salmonid alphavirus (SAV) in insect cells and obtained functional processing and secretion (Metz et al., 2011). They recently developed such a process to obtain CHIKV VLPs within chimeric baculovirus. The first vaccination assays and virus challenge experiments were performed using the A Suhrbier mouse model system (Metz and Pijlman, 2011, Pijlman, 2012).

7.3. Vaccination with chimeric alphaviruses

Chimeric alphaviruses have been developed containing Venezuelan equine encephalitis virus (VEEV) or equine encephalitis virus (EEV) nonstructural protein coding sequences and CHIKV structural protein coding sequence with a programmed, attenuated, cell-type-restricted phenotype because of the lack of Old World alphavirus nsP2 or New World alphavirus capsid involved in innate cell response shutdown (Kim et al., 2011). To make these viruses incapable of transmission by mosquito vectors and to differentially regulate the expression of viral structural proteins, their replication was made dependent on the internal ribosome entry sites (IRES), derived from the encephalomyocarditis virus IRES (Plante et al., 2011).

The design of the genomes was complemented by selection procedures, which adapted viruses to replication in tissue culture and produced variants which (i) demonstrated different levels of replication and production of the individual structural proteins, (ii) efficiently induced the antiviral response in infected cells, (iii) could not replicate in mosquito cells (Darwin et al., 2011), and (iv) efficiently replicated in Vero cells. Finally, this vaccine candidate was tested in highly sensitive A129 mice (IFN-α/β R−/−) and was found not deleterious in contrast to CHIK 181/Clone 25 vaccine strain but efficiently induced a protective immune response (Wang et al., 2011b) and was able to provide cross-protection for alphaviruses of the Semliki Forest antigenic complex (Partidos et al., 2012).

Using the same procedure and CHIKV vaccine candidate, Partidos et al. induced a cross-protective immunity in adult AG129 mice (IFN-α/β R−/−; IFNγ R−/−) against ONNV 9 weeks post vaccine (Partidos et al., 2012). The authors also showed that transplacental passive anti-CHIKV antibody transfer to 3 week old A129 offspring from the vaccinated dam was protective against a ONNV intra-dermal challenge.

Using a completely different strategy, GenPhar developed a recombinant adenovirus CadVax-CHIKV containing codon optimised sequence containing the CHIKV structural genes (C–E3–E2–6k–E1). This vaccine was tested using the A Suhrbier mouse model (Wang et al., 2011a). At 5 weeks post-vaccination, the reciprocal of the neutralisation titre (RNT) was close to 5 × 10³ which was similar to the titre observed after infection with the wild type CHIKV (100% protection against cytopathic effect of 200 CCID₅₀ of virus). The mice were challenged 45 days after vaccination and were shown to be fully protected against both foot swelling and viral replication (undetectable compared to 5 × 10⁶ CCID₅₀ in control group).

Other recombinant viruses tested in mice include chimeric vesiculo/alphavirus, in which the entire CHIKV envelope polyprotein was expressed (E3–E2–6k–E1) (Chattopadhyay et al., 2013). These VSV-G-CHIKV chimeras were attenuated in tissue culture, propagated to high titre without VSV-G complementation and shown to protect adult mice C57BL/6 challenged 34 days postvaccination by the sc route of inoculation in the left rear foot-pad (Morrison model). Efficiency of vaccination was assessed for neutralising antibodies using the plaque reduction neutralisation test PRNT80 160 to >640 and IFN-γ ELISPOT assay against E1 and E2 peptides (400–600 ± 200 SFC/10⁶  cells). However, in this study the viral replication and disease induced by the CHIKV in non-vaccinated mice was not very high (2.85 ± 0.47 log10 pfu/ml at day 2), i.e., 2–3 orders of magnitude below the value obtained in two others studies (Gardner et al., 2010, Teng et al., 2012), perhaps because of a shorter delay between vaccination and challenge.

7.4. Electroporated DNA

A plasmid containing codon and RNA-structure-optimised E3–E2–E1 protein sequences without the 6k peptide but including the furin cleavage sites as well as a Kozak-IgE leader sequence at the 5′ end of the genome, was generated by GeneArt (Regensburg, Germany) and vaccination was performed by 3 subsequent electroporations into quadriceps from 8-week-old female BALB/c mice (Mallilankaraman et al., 2011). One week after the third electroporation CHIKV neutralising antibodies were significantly enhanced with a RNT median of 320 (which produced 100% protection against cytopathic effect using a challenge dose of 100 CCID50 of virus) and a high CHIKV specific cellular response was demonstrated as measured by IFN-γ ELISPOT assay against E1 and E2 peptide pools (1613 ± 170 SFC/10⁶  cells). This construction thus induced an improved immune response, when compared with the previous C–E1–E2 contruction from the same group (Muthumani et al., 2008). At this time mice were challenged by intranasal exposure with 107 pfu (25 μl) of the CHIKV-PC-08 strain (ECSA genotype).

In summary, the mice were protected from the high morbidity normally associated with intranasal infection (i.e., spongiform lesion apoptotic bodies, microglial nodules in the external granular layer of the cerebral cortex; severe myocardial and liver degeneration/necrosis) which required euthanasia of control mice at day 6–12 pi. Despite marked weight loss during the first 3 days, vaccinated mice returned to normal values thereafter. However, in the vaccinated animals sacrified at day 5 pi, some microglial oedema was seen in the brains suggesting that immunisation decreased CHIKV-mediated pathology but did not fully protect the brain in the intranasal inoculation model. In contrast, the disease in mice vaccinated with DNA expressing only the CHIKV capsid induced a morbidity picture identical to the control non-immunised mice. Finally Mallilankaraman et al., 2011 tested the immune response induced following this vaccination strategy in four rhesus macaques (5 immunizations). Two weeks later, they detected variable neutralising antibody responses (RNT 80–1200) and specific T cell responses (200–500 SFC/10⁶  PBMCs), but they did not challenge the animals. Thus, all candidate vaccines except one (TSI-GSD-218) have so far only been tested in mice, and the studies have mainly focused on neutralising antibodies and not on cellular responses. From the literature, it is unclear whether any of these vaccines have proceeded into human clinical trials.

In addition, recent studies have shown that (1) the recognised protective immune response against CHIKV consists predominantly of IgG3 antibodies and (2) the early neutralising IgG response to CHIKV in infected patients targets a dominant linear epitope on the E2 glycoprotein (Kam et al., 2012a, Kam et al., 2012b). However, another study reported that CHIKV is able to escape the impact of neutralising antibodies by direct cell-to-cell transmission (Lee et al., 2011). This observation implies that an effective vaccine cannot be assessed purely by its ability to induce detectable neutralising antibodies.

In conclusion, whilst it is evident that an effective vaccine against CHIKV infection is clearly needed, inactivated vaccines are unlikely to provide strong and long-lasting immunity, and they will almost inevitably require multiple doses, thus reducing their cost effectiveness. DNA vaccines, thus far, have proved less effective in humans than in other mammals. Live, attenuated alphaviruses are being used as the genetic backbone for vaccines under development by Chiron, Alphavax and others and this approach is the most likely to lead to an effective vaccine with long-lasting immunity for CHIKV. However, it remains to be determined whether the inevitable high development costs of a safe and effective live, attenuated chimaeric vaccine against CHIKV will prove to be a restraining influence.

8.1. Experimental therapies targeting steps in viral replication

The authors are grateful to Dr Christian Devaux for providing access to figures from Solignat et al. (2009), Dr Jean-Claude Desenclos and Dr Harold Noël (Institut de Veille Sanitaire, France) for providing unpublished data about French imported cases and Pr Fabrice Simon for helpful discussion. LDM is a post-doc financed by the ICRES Project of the European Commission.