The Chandipura Encephalitis

Chandipura Virus (CHPV) infection is one of the most recent and unattended class of diseases in India; originally an arthropod born virus (Arbovirus) CHPV endemic to India only [1]. It was discovered in 1966 by Bhatt and Rodriguez, at Virus Research Centre (VRC), Pune. It was discovered in patients suffering from febrile illness in Chandipura village in northern Maharashtra near Nagpur district, during an outburst of Chikungunya and Dengue. The virus draws its name from the area where it was first isolated [2] [3].
CHPV has been implicated in several encephalitic outbreaks that have occurred in various districts of Andhra Pradesh (AP), Maharashtra and Gujarat during the past few years. This virus was not considered to have an epidemic potential until an outbreak of acute encephalitis in children (9 months to 15 year of age) in Andhra Pradesh, India was attributed to CHPV in 2003 with high case fatality rate (CFR) of 55.6 percent [4]. During this outbreak, a case fatality rate of more than 55% was recorded. A similar outbreak of encephalitis in children was observed in Gujarat State in western India in 2004.

Figure 1: The three major affected areas of CHPV in India. Andhra Pradesh first reported a case in the year 1997, Maharashtra First reported a case in Chandipura (Nagpur) Village in 1965, Gujarat and near Madhya Pradesh first reported in 2004[c] [1][2][3].
In natural conditions, it has been isolated from a pool of 253 unidentified Phlebotomine sand flies (Phlebotomus spp.) in the Maharashtra, India and from unidentified Sergentomyia in the Karimnagar district in Andhra Pradesh, India. Four strains have also been isolated from batches of sand flies from Senegal, belonging to genus Sergentomyia [4][5]. It thus reveals that wide distribution of the CHPV and important transmitters include Phlebotomus and Sergentomyia the two genera of sand flies. Thus sandflies are the main vector of CHPV and are the maintenance host of CHPV [5].
1.1.1 Symptoms and Pathology
The symptoms of CHPV starts as an influenza-like illness, associated with abdominal pain, vomiting, altered consciousness, and impaired neurologic functions [6]. It causes encephalitis, a diffuse or focal inflammatory process of the brain parenchyma associated with brain dysfunction.
The pathology of encephalitis can be broadly divided into two major categories:
(i) Infection-related encephalitis
(ii) Auto-immune mediated encephalitis
Infection related encephalitis is a direct result of viral entry into the Central Nervous System (CNS), whereas auto-immune mediated encephalitis is linked with systemic infection or vaccination and is mediated by a pathologic immune response mainly directed against myelin and affects the brain and the spinal cord. Arbovirus borne diseases are symptomatically treated as no specific treatments are available [7].

1.1.2 Diagnosis
With the emergence of this pathogenic condition it has become necessary to provide early and sensitive diagnosis of this disease. Evaluation of potential candidate vaccine(s) and antiviral treatment strategies, sensitive and specific viral quantitation assays are necessary need of the hour. The general diagnosis is made on lines similar to encephalitis; a computed tomography scan (CT scan) of a child brain with CHPV infection. The right side of the brain shows abnormal dilation of the ventricles [8].

The various diagnostics being reported and being used for providing diagnosis for management and control of disease are:
Detection or Tests Techniques Involved for Diagnosis
Virus Isolation Cell Culture, Infant mice, Embryonated eggs [in ovo (eggs)]
Antigen Detection Enzyme Linked Immunosorbent Assay (ELISA), Real Time PCR (qPCR) to detect and quantitate Viral load, PCR of G gene, Immunofluorescence Assay (IFA), detection with nested PCR.
Serological Test IgM and IgG ELISA.
Table 1: Tests for detection of CHPV
It has been reported that Real time one step RT-PCR assays offers an excellent alternative with several advantages such as high sensitivity, speed, accuracy and reproducibility.
1.2 Incidence of the Disease
CHPV is a relatively new albeit strong player in the human pathogenic domain. It has started garnering attention only recently due to the steadily increasing number of cases of encephalitis over the past few years and the equally high proportion of mortality of infected patients. Thus, comparatively less information is available about the virus and its mode of transmission. It has also been observed that this virus shares marked similarities with the prototype virus of the Rhabdoviridae family Vesicular Stomatitis Virus (VSV) particularly notorious for infecting cattle, horses and pigs in different parts of the world. Also CHPV has many parallels with the Rabies virus which again belongs to the same family [9].
CHPV belongs to the order Mononegavirales and Rhabdoviridae family. CHPV was first isolated from the village of Chandipura in Maharashtra during an outbreak of Chikungunya and Dengue in 1965. In addition to being dangerous and fatal, CHPV is also endemic to India which only increases its importance pertaining to research and characterization. CHPV has been implicated in several encephalitic outbreaks that have occurred in occurred in various districts of Andhra Pradesh (AP), Maharashtra and Gujarat during the past few years. These outbreaks were unique because of the fact that the disease progressed rapidly and killed children below the age of 15 yr within 48 h of hospitalization; with a case fatality rate of more than 55 and 75 per cent in AP and Gujarat respectively [9].
Figure 2: Impact of CHPV and Vesicular Stomatitis Viruses all over the world[8].
1.3 Chandipura Virus
Chandipura Virus (CHPV) is a bullet shaped enveloped RNA protein virus having the genome size of approximately 11 kb. CHPV belongs to the taxonomic group of numerous related virus order Mononegavirales. All the viruses belonging to this family share some typical features. These viruses possess a linear, non-segmented, single-stranded, RNA of negative polarity which produces 5-10 distinct mRNAs from a single promoter located in the 3' end of the genome. Another major feature of this order is that all the viruses belonging to all the families in this order encode for an RNA-dependent RNA polymerase (RdRp) that is highly homologous to those of other mononegaviruses[10][11].
The CHPV belongs to the Rhabdoviridae family. This name is derived from Greek rhabdos meaning rod referring to the shape of the viral particles. All these viruses are typically rod shaped or bullet shaped. One of the main features of this family is that Rhabdoviruses infect a broad range of hosts throughout the animal and plant kingdoms. Rhabdoviruses infect insects, fish, and mammals including humans [11].

Figure 3: Figure showing the structure of CHPV [5].
1.3.1 Virus Life Cycle
The life cycle of CHPV can be segregated into various distinct stages. They include adsorption of virus particle onto the host cell, penetration of virus into cell, uncoating and release of core RNP into the cytosol from late endosomal vesicles, transcription of the genome by viral polymerase, translation of viral mRNA, post-translational modifications of viral proteins, replication of viral genome, assembly of progeny particles and finally budding of mature virion. It is very important to notice that that the entire lifecycle of the virus in its host is cytosolic and at no point does it enter the nucleus of the infected cells [12]. The encapsidated negative-stranded RNA serves as a template for transcription by the polymerase complex i.e. the viral RdRp composed of L and P proteins from the 3' end of the genome. Sequential transcription starts with the formation of a short uncapped leader RNA (lRNA) from the 3' end followed by the formation of 5 monocistronic, capped and polyadenylated viral mRNAs. Once the 5' end-capping and polyadenylation is accomplished, translation of the viral mRNAS takes place using the host machinery which leads to the accumulation of viral polypeptides within the infected cells and this acts as a trigger for RdRp to switch from transcription mode to replicative mode and it copies the entire genome into an exact polycistronic complement that acts as replication intermediate to produce many more copies of negative sense genomes upon further rounds of replication [13].
The entire vesiculovirus life cycle within an infected cell is cytosolic. Genome RNA enwrapped with nucleocapsid protein N acts as a template for sequential transcription starting from 3' end of the genome to synthesize short leader RNA and five monocistronic capped and poly adenylated viral mRNAs. Viral RNA dependent RNA polymerase (RdRp) is composed of large protein L, the catalytic subunit and phosphorylated form of Phosphoprotein P that acts as a transcriptional activator. Translation of viral mRNAs results in accumulation of viral polypeptides within infected cells and set up stage for the onset of genome replication [14].
Figure 4: Life Cycle of CHPV [6].

1.3.2 Genome organization of CHPV
CHPV genome RNA comprises of a 49 nucleotide long leader gene (l), followed by five transcriptional units coding for viral polypeptides separated by intragenic spacer regions and a short non-transcribed 46 nucleotide long trailer sequence (t) arranged in the order 3' l-N-P-M-G-L-t 5'. These genes typically codes for five proteins: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M). Rhabdoviruses that infect vertebrates are usually bullet-shaped [15].
Figure 5: Figure showing the genome organization of CHPV.[16]
The functional and structural information of the CHPV proteins has been extended from the research literature on the prototype virus of the genus Vesicular Stomatitis Virus (VSV). Nucleocapsid protein encapsidates the viral RNA genome and forms the Nucleocapsid-RNA (N-RNA) which protects the RNA from degradation during various stages of viral life cycle. N-RNA is present in association with viral RNA dependent RNA polymerase (RdRp) which is composed of L protein the catalytic subunit and phosphorylated P protein (the transcriptional activator). The complex of L, P and N proteins along with RNA is known as ribonucleoprotein (RNP) particle [16]. P protein is the regulatory protein responsible for the switch between transcription and replication. During virus assembly, the M protein interacts with the ribonucleoprotein particle and condenses it into a tightly packed helix, giving the virus its characteristic bullet shaped morphology. The M protein associated RNP is surrounded by a lipoprotein envelope that contains trimers of G protein spiking out from the virion surface. G protein is a single pass type I transmembrane protein, which has a short cytoplasmic tail, a transmembrane domain and an antigenic ecto domain. It is the only spike protein of CHPV that assists virus adsorption, assembly and budding. Viral entry, a critical step in the life cycle of the virus, is mediated by G protein.

In addition to its structural role, Matrix (M) protein balances virus transcription and replication by condensation of nucleocapsid in tightly coiled helix. It has been shown to be responsible for directing the virus budding from infected host cells. It inhibits the mRNA nuclear export by directly interacting with host factors, hence shutting off cellular transcription. M has also been known to induce cell rounding and apoptosis in infected cells, hence responsible for the cytopathic effects [16].
1.4 G- Protein
The study of the G-protein forms the crux of our major project. We aim at determining the viral and host interactions that facilitate the entry of Chandipura into the host cells and trigger the infective mechanisms behind the encephalitis in the infected patients. It has long been proven that the G-protein forms the first point of interaction between the host and viral cell; and hence it has also been proposed that curtailing this interaction between the two cells can prove as an effective method for prevention and abatement of this disease. It is the only spike protein of CHPV that assists virus adsorption, assembly and budding of the virus. Viral entry, a critical step in the life cycle of the virus, is mediated by G protein. This study mainly centers around the ecto domain of the G-protein which is essentially the entire G-protein with transmembrane domain removed so that it is available in soluble form and it is easier to study the expression profile and interactions of G-protein with the host proteins [17].
Protein Functions
N - Protein ' Forms the core nucleocapsid by enwrapping the genomic RNA.
' Associated with viral RdRp.
P - Protein ' Acts as transcriptional activator in its phosphorylated form.
' In unphosphorylated state it is found in association with N protein.
' Acts as a switch between viral replication and transcription.
M - Protein ' Present on the inner surface of virion.
' Tethers core nucleocapsid to the membrane.
' It plays both structural and functional role in the life cycle of the virus and is mainly responsible for cytopathic effects of the viral infection.
G - Protein ' Present on the surface as the spike protein.
' Mediates viral entry into the host cell.
' Helps in virus adsorption, assembly and budding.
' Acts as an antigenic determinant.
L - Protein ' Responsible for catalytic activity of RdRp.
' Major functional and catalytic subunit of the RdRp.
Table 2: Functions of Virus structural Proteins
1.5 Probable Host Protein interactions of CHPV G-protein
1. Protein Phosphatase-1 Regulatory Subunit(PPP1R3F)

' Subcellular location: membrane, single-pass membrane protein.
' Function: glycogen-targeting subunit for protein phosphatase1(PP1).
' Tissue specificity: expressed in brain, skeleton muscle & heart.
' Biological function: regulation of glycogen synthase(GS) activity.
' Hypothesis: R3F, a novel membrane-associated glycogen targeting subunit of protein phosphatase 1 regulates glycogen synthase in astrocytoma cells in response to glucose and extracellular signals. Glycogen is the only store of glucose in brain i.e, only energy source in brain/astrocytoma cells. Synthesis and degradation of glycogen in brain is regulated by an enzyme GS (glycogen synthase). Key regulator of glycogen levels is glycogen targeted protein phosphatase1(PP1). PP1 dephosphorylates and activates glycogen synthase (GS) leading to an increase in glycogen synthesis[22].
In absence of glucose, phosphorylation of PP1R3F, facilitates glycogen degradation and provide glucose to astrocytoma cell. In brain, glycogen is the only energy source whose synthesis is regulated by PP1 with its glycogen binding protein. R3F present at high level in the rodent brain. R3F binds to PP1 through a classical RVXF binding motif. R3F binds to glycogen as well as to membrane also. R3F has the affinity towards membrane also.
In glucose deprivation state, GS interacts with PP1-R3F that leads to increase the phosphorylation of PP1-R3F bound GS, it reduces glycogen synthesis and facilitates glycogen degradation to provide glucose in astrocytoma cell.
Protein phosphatase 1 regulates the histone code for long term memory. In the adult brain, PPs are essential for synaptic function and are involved in the negative regulation of higher order brain functions such as learning and memory. Protein phosphatase 1 (PP1) is a critical regulator of chromatin remodeling in the mammalian brain that controls histone PTMs and gene transcription associated with long-term memory. Memory loss is one of the symptom of CHPV infection in patients before death[22].
' Inhibition of nuclear pool of PP1 should alter histone post translation modification in neurons and have an impact on gene transcription associated with long term memory. If the virus take over the control on PP1 then it will not lead to continue the glycogen synthesis in brain and ultimately leads to apoptosis.

2. Galactose 3-O-Sulfotransferse4 (GAL3ST4)

' Subcellular location: Golgi apparatus (Golgi stack membrane); single pass type II membrane protein.
' Function: It catalysis the transfer of sulfate to beta- 1,3 linked galactose residues in O- linked glycoproteins.
' Expression: Expressed mainly in placenta, testis, ovary, spinal cord, brain and adrenal glands.

' Hypothesis: Sulfotransferases mediated sulfonation is an essential step in promoting activity of the Proteoglycans in the host cells. This critical step occurs in the golgi apparatus and leads to the activation of Proteoglycans. Proteoglycans play several significant roles in maintaining the homeostasis of the host cells and also in various signaling pathways. Thus interaction of GAL3ST4 may hinder this modification in host, curtailing the signaling pathways. Also, since it is a transmembrane protein it can be used by virus to enter into the host cells. Also GAL3ST4 helps in the sulfonation of Heparan sulfate; which acts as cementing material between two adjacent cells and helps in the transfer of signaling molecules. Thus interaction of virus with this enzyme can severely affect the viability of the cell by inhibiting various signaling pathways [23].

3. Actin Beta(ACTB)

' Subcellular location:- It is localized in Nucleus, Cytoplasm and cytoskeleton. Localized in cytoplasmic mRNA granules containing untranslated mRNAs.
' Function:- Actin is highly conserved proteins it forms cytoskeleton and helps in virus entry into the host cell.
' Expression:- It is expressed in almost every cell of the body.

' Hypothesis:- The ACTB gene provides instructions for making a protein called beta (??)-actin, which is part of the actin protein family. Proteins in this family are organized into a network of fibers called the actin cytoskeleton, which makes up the structural framework inside cells. There are six types of actin, four are present only in muscle cells, where they are involved in muscle contraction. The other two actin proteins, ??-actin and gamma (??)-actin (produced from the ACTG1 gene), are found in cells throughout the body. These proteins play important roles in determining cell shape and controlling cell movement. Studies suggest that ??-actin may also be involved in relaying chemical signals within cells. The endocytosis of bullet-shaped Rhabdoviruses, due to their larger size, requires the actin cytoskeletal machinery. This machinery is engaged during endocytosis after the coat formation, which triggers membrane deformation and internalization, CHPV G- protein in the present study has been identified to interact with different types of actin (ACTA1, ACTB, and ACTC1) and Dynamin (DNM1 and DNM2) proteins that are known to aid in the clathrin-mediated endocytosis of Rhabdoviruses [25].

4. Dolichyl-Diphosphooligosaccharide-Protein Glycosyl Transferase (DDOST)
' Subcellular location: It is localized on the Endoplasmic reticulum membrane; Single-pass type III membrane protein. The single transmembrane helix has a kink in the middle of the transmembrane span.
' Function: It catalyzes the transfer of high mannose oligosaccharide (GlcNac2Man9Glc3) onto asparagine residues (or within Asn-X-Ser/Thr consensus motif) of nascent polypeptides as they enter the lumen of the rough endoplasmic reticulum.
' Expression: It is expressed in all tissues, expressed extensively in heart.

' Hypothesis: Since Asparagine linked glycosylation of proteins is a ubiquitous post translation modification reaction in eukaryotic organism that occurs in RER. CHPV interacts with RER to undergo translation and further the synthesized proteins are modified by N-linked glycosylation at two positions in G protein. Thus interaction of CHPV G-protein with DDOST is to mediate glycosylationof its proteins to generate an active virus particle. It can also be hypothesized that interaction may hinder or inhibit this post translational modification of proteins of affected individual. Thus it can be a probable virus host interaction [29].

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