Abstract
Background: Chikungunya Virus is a single stranded RNA virus of the genus alphavirus. The transmission of this virus occurs
by the bite of infected mosquitoes. It is characterized by an abrupt onset of fever frequently accompanied by joint pain.
Aim: Molecular Characterization of Chikungunya Virus in Serum- Relevance for Disease Management.
Methods and Materials: 30 cases were considered having symptom of fever suspected to have chikungunya. RNA was
isolated from magnetic beads methods which were further studied for Real Time-PCR (RT-PCR).
Results: Among the 30 cases suspected for chikungunya fever,
only 18 were positive for the same. Hence, the prevalence was
calculated to be 60%. About 39% of the positive cases were in the age
range of 21-40 years, which is the active age group of the
total positive cases, 66.66% were males and 33.33% were females. The
virus can be even transmitted together as a co-infection.
Patients infected with chikunguniya virus are more likely to experience
symptoms of high fever, severe polyarthralgia and rash with
lymphopenia.
Keywords: Chikunguniya; Real time polymerase chain reaction; Aedes aegypti; Aedes albopictus
Abbrevations: CHIK V: Chikungunya Virus; CMRL: Central Molecular Research Laboratory; SGRRIM & HS: Shri Guru Ram Rai
Institute of Medical & Health Science; CDNA: Complementary DNA; MRNA: Messenger RNA
Introduction
Chikungunya Virus (CHIKV) is an alphavirus with a positive
sense single-stranded RNA genome of approximately 11.6kb, 60-
70nm diameter capsid and a phospholipid envelope. It is sensitive
to desiccation and to temperatures above 58°C. It is a member of
the Semliki Forest Virus complex. The transmission of this virus
occurs with the bite of infected mosquitoes of the genus Aedes. In
addition, blood born transmission is possible [1-3]. The incubation
period ranges from 3 to 12 days. The onset is usually abrupt and
the acute stage is characterized by sudden high fever, incapacitating
arthralgia, myalgias and skin rash. Chronic arthritis may develop in
the patients and is associated with fever, asthenia and exacerbation
of arthralgia, inflammatory polyarthritis, and stiffness [4,5]. The
Alphavirus group comprises of 28 viruses, six of which can cause
human joint disorders-namely chikungunya virus, o’nyong-nyong virus (central Africa), Ross River and Barmah Forest viruses
(Australia and the Pacific), Sindbis virus (cosmopolitan), and
Mayaro virus (South America, French Guyana). These alphaviruses
share certain antigenic determinants [6-9].
Materials and Methods
30 clinical samples were considered in this study. Blood samples
were taken from the suspected cases of Chikungunya from the
different departments of Shri mahant Indiresh Hospital, dehradun
(Uttarakhand) and further processed at Central Molecular Research
Laboratory (CMRL), Shri Guru Ram Rai Institute of Medical &
Health science (SGRRIM & HS), Patel nagar, dehradun (U.K.) for the
molecular characterization of Chikungunya Virus.
Collection of EDTA Blood & Separation of Serum/Plasma
Whole blood samples were centrifuged at 5,000rpm for 10-
15 minutes at 4°C. The serum was separated from blood & then
subjected for nucleic acid extraction. Then Isolation of RNA for
the detection of chikungunya virus was done by magnetic beads
method where Magnetic carriers bearing an immobilized affinity or
hydrophobic ligand or ion exchange groups or magnetic biopolymer
particles having affinity to the isolated structure are mixed with the
sample containing target compounds. The specimens with target
nucleic acid are processed by lysis buffer and then the nucleic acids
are efficiently bound to the specifically modified magnetic beads.
The beads were removed from the solution and attached to the tube
wall manually using a magnetic separator stand. After washing,
purification and elution, ultra-purified RNA can be obtained. After
RNA was obtained master mix was prepared for chikungunya RNA
for qualitative detection by Real Time PCR. Reverse transcription
was done (RT-PCR) for the extracted RNA specimens. In this method,
RNA was first transcribed into complementary DNA (cDNA) by
reverse transcriptase from total RNA or messenger RNA (mRNA).
The cDNA is then used as the template for the qPCR reaction.
Results
Among the 30 cases suspected for chikungunya fever, only 18
were positive for the same. Hence, the prevalence was calculated
to be 60%. About 39% of the positive cases were in the age group
ranging in between 21-40 years, which is the active age group of the
total positive cases, 66.66% were males and 33.33% were females.
Ct value in molecular profiling indicates that the sample is positive
for chikungunya viral genome whereas no Ct value is indication of
negative results for chikungunya. Ct value for internal control was
in the range of 14-17 shows that the result is valid (Figure 1).
Figure 1: Amplification curves in Real time PCR for Chikungunya virus.
Discussion and Conclusion
Chikungunya (CHIKV) is a mosquito-borne disease caused by
an RNA alphavirus of the Togaviridae family. The main mosquito
vectors are the aggressive Aedesaegypti and Aedesalbopictus. The
most common presentation of CHIKV is acute onset of fever and
polyarthralgia, sometimes followed by a macupopular rash. Other
associated symptoms can include headache, myalgia, nausea and
vomiting. These symptoms typically occur 3 to 7 days after the
mosquito bite and generally last 7 to 10 days. Rare complication of
CHIKV can include uveitis, retinitis, myocarditis, hepatitis, nephritis,
bullos skin lesions, hemorrhage, meningoencephalitis. The clinical
manifestations of CHIKV can be very similar to dengue fever, which
is transmitted by the same species of the mosquito. The 2 viruses
can be even transmitted together as a co-infection. Patients infected
with CHIK are more likely to experience symptoms of high fever,
severe polyarthralgia and rash with lymphopenia, whereas patients
with dengue fever are more likely to have symptoms of hemorrhage,
shock and death with associated laboratory derangements of
neutropenia and thrombocytopenia. However, because of the
close similarities, dengue fever should be strongly considered
in the differential diagnosis of patients suspected with CHIK.
Conformational testing for CHIK can be performed in one of the three
ways. These methods include viral culture if tested within first three
days of illness, PCR in the first eight days, or antibody serology after
the first three days of illness. Other laboratory abnormalities may
include lymphopenia, thrombocytopenia, elevated creatinine and
elevated hepatic transaminases. Treatment is primarily supportive.
The preventive measure among the individual level included the use of
mosquito repellents like coils, body creams, mosquito nets,
etc. At the community level, it is important to ensure that there are
no collections of water in household vessels or around dwelling
places. The usual recommendation is that on every fifth day, all the
vessels, which contain water, should be emptied; this observance of
a dry day breaks the life cycle of the mosquito [10].
From above discussion, we conclude that, Chikungunya fever
is self-limiting, the morbidity can be very high in major outbreaks,
resulting in heavy social and economic tolls. The prevention of
the disease requires a planned approach, besides knowledge
and awareness on the early warning signs. An integrated vector
management through the elimination of the breeding sites, the
use of anti-adult and anti-larval measures and personal protection
will contribute to the prevention of outbreaks. A community
empowerment and mobilization is crucial for the prevention and
control of Chikungunya.
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