Introductory Chapter: Introduction to Chikungunya

*Jean Engohang-Ndong*

#### **1. Introduction to chikungunya**

The "Golden Age of Microbiology" that spanned from roughly 1857 through 1914 was not only marked by the identification of many bacterial infectious agents that ravaged the lives of millions of people across the world with *Mycobacterium tuberculosis*, the causative agent of human tuberculosis being one of the most dreaded infectious bacteria discovered at the time, that period was also very special because the work on viruses started. Indeed, in 1892, Dmitri Ivanosky discovered that the sap of diseased tobacco plants could infect healthy tobacco plants even after passing the sap through a Chamberland's porcelain filter which was typically used to trap bacteria. While Dmitri hypothesized that his results could be the consequence of cracked filters, Martinus Beijerinck demonstrated in 1898 that what he called *Contagium vivum fluidum* (contagious living fluid in Latin) contained infectious particles which size was much smaller than bacteria in such a way that they could persist in the filtrate through the Chamberland's porcelain filters. Later, the discovery and partial characterization of bacteriophages opened new avenues for the explosion of the study of viruses.

The invention of the electron microscope in 1931 revolutionized the study of viruses when for the first time, bacteriophages were shown to have a very complex structural organization. As microscopy evolved to unravel the tremendous diversity of the viral world, the progress in molecular biology, biochemistry, and biophysics allowed the development of a structural model that led to the characterization of not only bacteriophages and plant viruses, but also animal viruses including the influenza virus, the poliovirus, and the Epstein-Barr virus to name only those.

The second half of the 20th century marked the discovery of over 2000 bacterial, plant, and animal viruses. Among the human virus newly discovered was the chikungunya virus (CHIKV) which is the etiologic agent of the chikungunya fever (CHIKF) and was discovered in Tanzania in the 1950s [1]. CHIKV which belongs to the *Togaviridae* family and to the genus of *Alphavirus*, is known to cause fever and joint pain that wear off within a week in most patients. However, in some patients, the joint pain may linger for months and/or eventually lead to arthralgia. Other symptoms may include headaches, myalgia also known as muscle pain, joint swelling, or rash. The next few paragraphs of this chapter and subsequent chapters of this book will explore the organization of the virus, the history of the disease it causes, its epidemiology, the current treatment status, and perspectives for new treatment and prevention of the disease.

#### **2. Genomic and structural organization of the chikungunya virus**

Since its discovery in the 1950s, CHIKV has been relatively well characterized, with a clear description of its genomic makeup and its structural organization (**Figure 1**).

#### **Figure 1.**

*Chikungunya virus genomic and structural organizations. (A) The genomic organization of CHIKV shows two groups of genes, including genes encoding non-structural proteins (NsP1-4) and genes encoding structural proteins C (capsid), E1 and E2 (envelope glycoproteins), and E3 and 6 K which are small accessory polypeptides. (B) 3D reconstruction of CHIKV showing the E1 basal triangle (red) and the E2 protrusion for each spike (green and yellow). (C) Shows spike-protein predicted structures based on atomic resolution structures of the envelope glycoproteins and high-resolution cryo-electron microscopic reconstructions of CHIKV [2, 3].*

Thus, like many other alphaviruses, full genome sequencing and cryo-electron microscopic reconstruction [2] revealed that CHIV is a small, spherical, enveloped virus with a genome of about 11.8 kb composed of a single-strand positive RNA flanked at its 5′- and 3′-extremities with untranslated regions (UTR). The role of these UTRs is to regulate important biological functions such as viral replication, transcription, and viral packaging during a lytic cycle. The 5′-UTR is preceded by a cap while the 3′-UTR is followed by a poly[A] tail. The cap and the poly[A] tail are both responsible for protecting the core information of the viral genome from the damaging effects of exonucleases. *In vitro* molecular and *in silico* analyses of the structural organization of the genome reveal that the CHIKV genome contains two open reading frames, ORF 1 and ORF 2 which are connected through a small polynucleotide sequence that is untranslated. That polynucleotide sequence constitutes a junction sequence (**Figure 1A**).

ORF 1 which is located at the 5′ end of the genome carries four genes which expression leads to the production of four non-structural proteins called NsP1, NsP2, NsP3, and NsP4. More specifically, CHIKV NsP1 is a viral capping enzyme while CHIKV NsP2 contains ATPase, RNA triphosphatase, helicase, and protease activities [4]. The two other CHIKV NsPs exhibit ADP-ribose-1-phosphate phosphatase and RNA-binding activities for NsP3 and RNA-dependent RNA-polymerase activity for CHIKV NsP4 [5]. These four non-structural proteins are essential for the replication of the viral genome during the infection cycle.

ORF 2 which is situated at the 3′ end of the genome arbores the five genes encoding five structural proteins including C, the capsid protein that forms a protective coat around the positive-RNA genome; E1 and E2, the two envelope glycoproteins, which both form the spikes on the surface of virion particles, and E3 and 6 K which are two small accessory proteins of the envelope. The viral spike proteins facilitate attachment to cell surfaces and entry of viral particles into the host cell.

*Introductory Chapter: Introduction to Chikungunya DOI: http://dx.doi.org/10.5772/intechopen.101892*

Using advanced imaging technology such as atomic resolution and cryo-electron microscopy, research teams have been able to reconstruct the three-dimensional organization of CHIKV and the fine structures of the viral envelope (**Figure 1B** and **C**). That imaging resolution of CHIKV shows that both E1 and E2 form a heterodimer in which both glycoproteins are intertwined and form the spikes on the surface of the virus. E1 and E2 have transmembrane domains that span the phospholipid bilayer of the viral membrane. The endodomains of both proteins interact with the capsid protein while the ectodomains of E1 and E2 that are also glycosylated make up the spikes which are suspected to be the receptor-binding domains responsible for attachment and entry of the virion into host cells [2, 3, 6].

### **3. Global threat**

It is now well established that since its discovery in Tanzania, CHIKV has been in circulation across the world and virtually in all continents including in Africa, in Asia, the Indian subcontinent, the Americas, and in Europe (**Figure 2**). It is thought that the virus circulated initially and primarily in sylvatic regions of sub-Saharan Africa in a cycle involving non-human primates and arboreal mosquito species of the *Aedes* genus as vectors. During the heavy rainy season, sylvatic mosquitoes multiply to the point that they would spread into villages and/or urban centers surrounding the rainforests where the arthropod could now infect human subjects. Following the enzootic sylvatic cycle and the human village cycle, the migration of villagers to urban centers and cities could lead to the spread of the infectious agents to humans living in these cities where the transmission cycle would now involve the urban mosquitos *Aedes aegypti* and/or *A. albopictus*. Spillover of mosquito vectors from villages to urbanized centers can also lead to the translocation of the infection to urban centers. Spillover infections have been documented in many African countries including, but not limited to Cameroon,

Senegal, South Africa, and Zimbabwe. From Africa, the chikungunya virus has now spread to Asia, Australia, Europe, India, and the Americas. In the Americas,

#### **Figure 2.**

*The spread of the chikungunya virus across the globe between the time of its discovery to the present [7].*

the disease has been found in southern states of the United States of America and in many countries of South America. Two major strains of the virus have been circulating in Africa: the eastern, central, southern African enzootic (ECSA) strain and the West African enzootic strain. Out of all the chikungunya virus strains found in circulation in the world, only ECSA and the west African strains involve a sylvatic cycle. Between 1958 and 1973, new CHIKV strains emerged as Asian strains in an urban cycle in many Asian countries, in India and a myriad of islands distributed between the south china sea and the Indian Ocean. In 2005, new outbreaks of the chikungunya fever emerged in the Indian ocean and in Asia. The CHIKV strain responsible for that outbreak was only involved in an urban cycle and never in a sylvatic cycle. Eventually, that Indian ocean strain spread to some parts of Africa and Europe including in France and Italy. More recently, the ECSA enzootic strain of CHIKV spread to South America while the Asian urban CHIKV strain spread to the two hemispheres of the American continent. Due to the ubiquity of air transport throughout the world, air travelers arriving from endemic areas to new territories contributed to the establishment and the spread of the chikungunya fever. Ultimately, new strains carried around the world including in Europe and in the Americas started circulating in local transmission.

The unusually dramatic increase in the incidence of the disease worldwide during the past decade could be explained by multiple factors including, but not limited to the improvement of transport means and increase of air travel which opened ways for a quick spread of the disease between continents; the expansion of urban areas in tropical regions of Africa and South America that were previously forested; the increased density in urban areas of human population and mosquito population of the genus *Aedes*; the new exposure of the of south Asian and the Indian basin populations to the vector of the virus. In addition to *A. aegypti*, since the mid-1980s, the invasion of the species *A. albopictus* which is known to be the major second CHIKV vector from Asia where it originated into surrounding continental countries and islands in the Indian Ocean basin, Africa, and southern Europe was made possible by a constantly growing global trade. Furthermore, the current increase in global temperature and subsequent projections of climate change will only exacerbate the spread of both *A. aegypti* and *A. albopictus*, therefore, a spread of the chikungunya virus to regions of the globe where the arthropod genus *Aedes* was not previously endemic. Taken altogether, these factors are creating perfect conditions for a CHIKV pandemic that is looming over the horizon.

In 2010, a genome-scale phylogenetic analysis of 40 CHIKV strains was performed to examine patterns of CHIKV evolution, and the origins of outbreaks recorded, as well as evolutionary rates that vary between enzootic and epidemic transmission. That study revealed that the CHIKV strains analyzed evolved from a common ancestor that existed within the last 500 years. The phylogenetic analysis also showed some geographic overlap between two main enzootic lineages that were previously thought to be geographically separated within the African continent. The study also estimated that CHIKV was introduced from Africa into Asia about 70–90 years ago. Based on the same study, the recent Indian Ocean and Indian subcontinent outbreaks appear to have emerged independently from continental East Africa [6].

#### **4. New perspectives**

The fight against the chikungunya virus has been arduous. However, despite that hard work and many accomplishments, a lot of work still remains to be done in many areas, including in prevention, diagnosis, and treatment of the disease.

#### **4.1 Prevention**

The golden approach to control an infection is to limit as much as possible exposure of sensitive subjects to the infectious agent. In the case of the chikungunya fever, a control of the proliferation of mosquito vectors and the development of a vaccine are two approaches the are being actively considered. In 2013, a team of researchers in Italy developed a cost-effective sterile insect technique with the final goal to suppress *A. albopictus* populations. *A. albopictus* is known to widely proliferate in man-made containers such as tires, buckets, and barrels with a high proportion of urban and suburban distribution. These two features made possible the application of the sterile insect control strategies. Thus, males *A. albopictus* were irradiated with gamma rays and released in the field. The adult population density was estimated by monitoring egg production in the ovitraps, while the radiation induced sterility was estimated by measuring the hatching percentage of weekly collected eggs in sterile insect technique and control sites. These experiments revealed that sterile males released at the rate of 896–1590 males per hectare per week led to a remarkable sterility level in the local population. Likewise, a high level of sterility led to an important decrease of the egg density in the ovitraps. Taken together, these two findings allowed researchers to formulate the assumption that if egg sterility values of at least 80% are achieved, that level should be enough to secure a suppression of the local population of mosquitoes [8]. While trials of control of mosquito populations using sterile insect technique is promising, it is nevertheless important to consider the immigration of mated females in the areas where sterilization of male mosquitoes was pursued. Therefore, combining sterilization of male mosquitoes along with an application of traditional population control methods such as the management of standing water or the application of insecticides is highly recommended.

In addition to innovative mosquito vector population control methods, a French company called Valneva SE recently developed a promising chikungunya vaccine to prevent the disease. In 2020, Valneva announced the initiation of a pivotal phase 3 clinical trial for its single-shot chikungunya vaccine candidate VLA1553. The promising chikungunya vaccine candidate VLA1553 is a live-attenuated CHIKV which was made mild by deleting a large part of the gene encoding the non-structural protein NsP3 [9]. This vaccine candidate was designed to prevent outbreaks and to provide protection against various CHIKV phylogroups and various CHIKV strains. The phase 3 clinical trial was conducted in the United States of America across 44 sites and involved 4115 adults which ages were 18 years and older. After a single shot, analysis of the serum of participants 28 days after inoculation of the candidate vaccine revealed that the vaccine induced a 98.5% protection in participants. Furthermore, when assessed for safety, results indicated that the CHIKV vaccine candidate VLA1553 was well tolerated by 3082 participants [10].

#### **4.2 Diagnosis**

Chikungunya fever, dengue fever, and the Zika fever are all caused by mosquitoes from the *Aedes* genus. The three diseases are characterized by an onset of fever, arthralgia, myalgia, and rash. That extreme similarity of symptoms between these different diseases leads most of the time and unfortunately to misdiagnosis and prescription of inappropriate treatment to patients. The lack of early accurate diagnosis of the disease may cause an evolution of the disease to complicated forms much more deleterious to the patient. The recent development of molecular diagnosis tools has opened new avenues for accurate diagnosis of the chikungunya fever and to tell it apart from the dengue virus infection or the Zika fever. The

primary laboratory test used to detect CHIKV or viral RNA is the analysis of the patient serum collected less than 6 days after the onset of the disease through a molecular biology approach called real-time reverse transcription polymerase chain reaction (RT-PCR). Real time RT-PCR is a laboratory technique of molecular biology commonly used for detecting the presence of specific genetic material of infectious agents including viruses. Originally, the RT-PCR technique used radioactive isotopes such as 32P to detect targeted genetic materials, but in the past two decades, the technique has been refined and radioisotopes have been progressively replaced by fluorescent dyes which are safer for the user and much more environmentally friendly. Real time RT-PCR allows researchers to see the results almost immediately while the reaction is still in progress. That approach was used by Bandeira and colleagues when they reported the presence of CHIKV RNA in the blood, urine, and semen during the acute phase of the disease in an adult patient with dengue virus (DENV) type 3 co-infection [11]. In that particular case, patient samples were first submitted to viral RNA extraction procedure followed by real time RT-PCR to screen for CHIKV, DENV, and the zika virus. Besides the real time RT-PCR technique, the second most reliable approach is by seeking CHIKV-specific immunoglobulin M (IgM) in patient samples. IgM detection is possible for sample collected roughly more than 5 days after the onset of the ailment [12, 13]. Thus, serological diagnosis makes it possible for physicians both experienced and young practitioners to draw a clear distinction between the chikungunya fever, the dengue fever, and the Zika virus infection and to develop an unambiguous diagnosis of the disease. It is worth noting however that the limited access to these two state-of-the art diagnosis approaches in remote rural, semi-urban, and urban endemic regions in countries where resources are not equally distributed still represents a huge obstacle to a systematic clear diagnosis of the chikungunya fever.

#### **4.3 Treatment**

To this day, there is no medicine approved by government agencies or international organizations such as the World Health Organization to treat CHIKV infection. Treatments commonly used during the course of CHIKV infection are typically geared toward the relief of symptoms such as fever and pain. Nonsteroidal anti-inflammatory drugs among which paracetamol, acetaminophen, and nibuprofen are often used to relieve symptoms of the disease. While not one single antiviral drug specifically directed against CHIKV has been approved, there are however several chemical components that have been investigated and stand as potential chemotherapeutic agents promising for the treatment of the chikungunya fever. Chloroquine for instance has shown to be effective against CHIKV through its obstructive activity against the entry of CHIKV into host cells [14]. Nevertheless, despite the promising *in vitro* results of that drug, clinical trials have failed to confirm any real benefit to patients. Similarly, suramin which has been approved by the Food and Drug Administration in the United State to treat trypanosomiasis which is caused by the protist *Trypanosoma brucei*, has also shown to be effective at preventing the entry of CHIKV in host cells during *in vitro* tests. Interestingly, while suramin is an effective anti-CHIKV drug *in vitro*, the adverse effects induced by the drug out weight the benefit to patients as the chikungunya fever remains mild in most patients. Multiple modified versions of suramin or suramin conjugates have shown promising results *in vivo* against CHIKV infection [15].

In addition to inhibitors of CHIV entry into the host cell, other promising anti-CHIKV have been identified and grouped based on their mode of action. Thus, some anti-CHIKV have shown to be NsP1 inhibitors, NsP2 inhibitors, and


#### **Table 1.**

*Selected promising anti-CHIKV compounds [14].*

NsP4 inhibitors while others exhibit an ability to inhibit the replication of the virus genome. Finally, some plant extracts harvested from the *Euphobiaceae* family with protein kinase C inhibitory activity also show strong anti-CHIKV activity (**Table 1**).

#### **5. Conclusion**

The chikungunya fever which is caused by a virus of the *Togaviridae* family, is characterized by the development of fever, and join pain. While the disease is rarely fetal, it can however evolve into severe arthralgia and myalgia. The development of modern molecular tools and atomic microscope tools have allowed scientists to achieve the sequencing and annotation of the virus complete genome and the resolution of the chikungunya virus structure. Thus, it is established that CHIKV is an enveloped virus with spikes that allow that bioparticle to attach to receptors located on the surface of host cells. CHIKV is not only occurring in tropical regions of Africa and south America, but it has also spread to other regions beyond the tropics in such way that it has become a global public health issue that requires a global approach for controlling the spread of the disease. The extreme similarity of symptoms found between CHIKV infection and some other viral infections including the Zika virus infection and the dengue virus infection has made it necessary to develop diagnosis tools specific for the detection of CHIKV infection in patients. Research interests in the development of vaccines and the discovery of anti-CHIKV drugs have opened the way to a strong vaccine candidate and to anti-CHIKV drug candidates. Nevertheless, the battle for controlling the chikungunya fever is far from being over.
