Chikungunya Virus (CHIKV) is a global public threat with its spread over 60 countries in Asia, Africa, Europe and the America. CHIKV is an alpha virus having 3 genotypes: Asian, West African and Eastern/Central/South African. It is an arbovirus which is small, spherical, enveloped, positive-stranded RNA virus. Since the receptors are present on the cells that develop cartilages, muscles and bones, and these cells are present inside the joints, CHIKV replicates inside the joints. According to the latest studies, Chikungunya occurs only once in a lifetime. The body develops protective antibodies that help avoiding the reoccurrence of the infection. It is not a fatal disease as its case mortality rate is 1 in 1000. Until today, there has been no licensed vaccine available for Chikungunya. The treatment is mainly focused in relieving of the symptoms. People over 65 years of age, neonates and the ones with chronic medical problems are most likely to have severe complications. Neonates are prone to vertical transmission of Chikungunya through their mother during delivery. Initial attempts for the development of CHIKV vaccine began in early 1960s. There are many antiviral drugs that have proven to inhibit the viral entry, replication or budding in model organisms but human. Many neutralizing antibodies have also been studied. There are variety of vaccines that have been researches like whole inactivated vaccine, CHIK-IRES, Chimeric vaccine, virus like particle vaccine etc but none of them have yet been FDA approved. The success of these vaccines depends on the safety, immunogenicity, stability and cost. The best method to prevent infection could be overall mosquito control as well as avoidance of mosquitos bites in the areas which are prone to Chikungunya.
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Introduction
Chikungunya is a mosquito-borne arthritogenic alphavirus which first out broke in Tanzania in 1952. Chikungunya in Kimakonde language refers to the ‘stooped/ contorted posture’ of the patient. It is transmitted by female Aedes mosquito vectors namely Aedes aegypti and Aedes albopictus. It has come into notice because of its emergence and spread over wide geographical areas causing massive epidemics. Symptoms of Chikungunya mainly include persistent fever and severe joint pain/ arthritis . These symptoms typically occur two to twelve days after the mosquito bite. Other symptoms may include headache, nausea, muscle pain, joint swelling, and a rash. Symptoms usually start improving within a week; however, sometimes the joint pain may last for months. Chikungunya has no cure and the treatment is focused on relieving of the symptoms. Chikungunya is episodic and epidemic in nature. Also, it has garnered attention due to difficulties in clinical diagnosis when ZIKA (ZIKV) and DENGUE (DENV) virus are simultaneously present.
1. MAJOR OUTBREAKS
1954-1968 : Philippines, Cambodia, Myanmar, Sri Lanka
1963-1973 : India
2005-2006 : Indian Ocean islands of Reunion and Mauritius
2006: India
2007: Italy
2016-2017 : Pakistan, India, Kenya
2. CHIKV
It belongs to genus Alphavirus and family Togaviridae. It is an arbovirus with a diameter of 700 Ao and is Icosahedral. In geometry, icosahedral refers to 20 faces.
2.1 STRUCTURE OF CHIKV
Chikungunya virus has 11.6 kb of positive sense single-stranded RNA genome. CHIKV has 5 structural and 4 non structural proteins. The structural proteins are capsid protein, glycoproteins E1, E2 ,E3 and 6K while nsP1-4 are non structural proteins. The glycoproteins E1 and p62 form heterodimers which subsequently trimerize to form viral spikes in the endoplasmic reticulum. During its transportation from acidic environment of Golgi to neutral environment of cell surface, p62 cleaves to form E2 and E3. E1 is involved in cell fusion and E2 thereby binds to the cell surface receptors. E3 facilitates the heterodimerisation of E1 and p62 and E3 releases after budding. E1 has 3 beta-sheets domains – I, II and III. A fusion loop is located at domain II tip. E2 has 3 Ig-like domains – A,B and C and a long domain D that connects domain B to C. Domain D interacts with E3 and fusion loop is sandwiched between A and B domains of E2. 3. SYMPTOMS
High Fever
Nausea
Fatigue
Severe Joint Pain
Headache
Maculopapular Rash
Polyarthralgia
Pain in abdomen
Pain at the back of the eyes
4. TRANSMISSION CYCLE
There are two transmission cycles for Chikungunya virus namely : Sylvatic cycle and Urban cycle. The sylvatic cycle exists primarily in Africa where the virus circulates between the forest dwelling Aedes and non human primates. When humans encroach these natural habitats, they are bitten by these mosquitoes thus causing a small outbreak. When these humans travel from rural areas to urban areas, they establish urban cycle. Urban cycle is basically a human-mosquito-human cycle.
5. LIFECYCLE OF CHIKV
Initial Stage of CHIV Infection
An infected female Anopheles mosquito introduces the virus into the bloodstream of the host. The CHIKV enters the human body through the salivary glands of the Anopheles mosquito. As soon as the virus enters the blood stream, the virus combines with permissive cells ( nose, throat and mouth).
Cellular and Infectious Stage
In this stage, the virus enters the cytoplasm and then the nucleus. In the nucleus, it deposits its genetic material and the genome is replicated inside the nucleus. After passing the cellular stage, the virus enters the neighboring tissues and starts infecting other cells. It proliferates within the bloodstream and the entire body and causes the infection. Symptoms occur 10-12 days post mosquito bite. Chikungunya is characterized by sudden onset of fever, severe joint pain and maculopapular rashes.
The Mosquito Stage
When a mosquito bites an infected human, the virus enters its body and replicates inside the mid-gut, neural tissues, ovary and fat. After the reproduction, it migrates to the salivary glands of the mosquito.
6. PATHOGENESIS
CHIKV replicates in the fibroblasts and synovial tissues. After the mosquito bite, the virus enters the human skin and replicates in the fibroblasts. On infection, it disseminates into the blood, liver, muscle and brain. When virus disseminates in the brain, it infects the choroid plexus of the stromal cells in the nervous system. This infection leads to the activation of innate immune response. Production of type I interferons is a primary response and is mainly involved against primary infections. Immune response to CHIKV involves production and activation of cytokines, chemokine and growth factors. During the acute phase, the levels of CXCL-9, CXCL-10, INF-α, IFN-γ and IL-6 increase and during the convalescent phase these level decrease. Increased level of IL-6 indicate inflammation and initiates adaptive T-cell response. CXCL-9 and CXCL-10 are involved in chemokine program and help in the migration of macrophages, monocytes, natural killer cells and memory T-cells. CCL-2 is referred to as Monocyte Chemostatic Protein – 1 (MCP-1) and leads memory T-cells and dendritic cells at the site of inflammation. Increased IL-6 levels cause joint pain. Also it leads to increased levels of CRP which in turn is involved in activation of Macrophage mediated phagocytosis. There is a condition known as lymphopenia ( low levels of lymphocytes). All of this causes low levels of monocytes and high viral load.
7. DIAGNOSTIC METHODS
While there are various methods for the diagnosis of Chikungunya, we have focused mainly on 2 methods namely, (i) IgM capture ELISA and (ii) Reverse Transcription-Loop Mediated Isothermal Amplification Assay (RT-LAMP).
7.1 IgM capture ELISA
It is a 2 step process where initially diluted samples are incubated in wells to allow binding of IgA, IgM and IgG to the specific antigens. Later, bound antibodies are are detected using a color catalyzing reaction which adds blue color to the positive ones. OD is measured at 450 nm and is compared to the standard one
This method is proven to be cost effective but it takes approximately 5 to 6 days for a person to develop antibodies.
7.2 Reverse Transcription Loop Mediated Isothermal Amplification Assay (RT-LAMP)
It uses reverse transcription method to synthesize cDNA from template RNA. For its amplification and detection, LAMP technology is used. Genomic viral RNA is the sample. Conserved 6K-E1(Envelope protein gene) region of CHIKV strains HD 180760 and STMWG01 are targeted. Oligonucleotide primers are designed from structural (E1) gene.
After mixing all these components and incubating at a constant temperature between 60o C to 65oC, amplification and detection is carried out via visualization through naked eye after addition of 1 milliliter of SYBR GREEN dye.
Results are studied as follows :
If Positive : Orange color changes to Green
If Negative : Orange color is retained
8. THERAPEUTICS
8.1 ANTIVIRALS
Antivirals basically act on specific stages of virus replication cycle by inhibiting viral entry, replication and budding. There are a variety of anti-CHIKV molecules that have been identified and studied. Some of which may include Favipiravir (T-705) which was identified in Japan and earlier was used to treat influenza virus and along with its de-fluorinated analogue T-1105, inhibited CHIKV replication in vitro. Further, another antiviral drug, Ribavirin, was identified which earlier was used to treat respiratory syncytial virus in infants, hepatitis C but later, in combination with interferon alpha, it tend to inhibit CHIKV replication in vitro.
An experiment was conducted in order to determine whether β-d-N4-Hydroxycytidine is capable of inhibiting virus replication or not. A number of experiments were conducted using 631 small molecules and β-d-N4-Hydroxycytidine was only molecule that inhibited replication over 80%.
PROCEDURE :
Renilla luciferase was added in density of 5000cells/well.
These were incubated at 37oC for 2 hours.
10 μM of NHC was added in triplicate.
DMSO was used for the negative control while rabivirin and favipiravin were used as positive control.
After 48 hours, effect of each of them was measured using Renilla luciferase assay kit.
Later, a time of addition experiment was conducted.
0.3 μM of NHC was added to Vero cell monolayer.
Samples were collected after 48 hours infection.
RESULT :
NHC was able to inhibit the viral replication at early stages.
There are several other drugs that have been identified as antiviral drugs effective on CHIKV infection in vitro and on model organisms but human. Therefore, more research is needed and is being done for the application of these novel drugs on infected humans.