Evidence of experimental vertical transmission of emerging novel ECSA genotype of Chikungunya Virus in Aedes aegypti.

BACKGROUND: Chikungunya virus (CHIKV) has emerged as one of the most important arboviruses of public health significance in the past decade. The virus is mainly maintained through human-mosquito-human cycle. Other routes of transmission and the mechanism of maintenance of the virus in nature are not clearly known. Vertical transmission may be a mechanism of sustaining the virus during inter-epidemic periods. Laboratory experiments were conducted to determine whether Aedes aegypti, a principal vector, is capable of vertically transmitting CHIKV or not. METHODOLOGY/PRINCIPAL FINDINGS: Female Ae. aegypti were orally infected with a novel ECSA genotype of CHIKV in the 2nd gonotrophic cycle. On day 10 post infection, a non-infectious blood meal was provided to obtain another cycle of eggs. Larvae and adults developed from the eggs obtained following both infectious and non-infectious blood meal were tested for the presence of CHIKV specific RNA through real time RT-PCR. The results revealed that the larvae and adults developed from eggs derived from the infectious blood meal (2nd gonotrophic cycle) were negative for CHIKV RNA. However, the larvae and adults developed after subsequent non-infectious blood meal (3rd gonotrophic cycle) were positive with minimum filial infection rates of 28.2 (1∶35.5) and 20.2 (1∶49.5) respectively. CONCLUSION/SIGNIFICANCE: This study is the first to confirm experimental vertical transmission of emerging novel ECSA genotype of CHIKV in Ae. aegypti from India, indicating the possibilities of occurrence of this phenomenon in nature. This evidence may have important consequence for survival of CHIKV during adverse climatic conditions and inter-epidemic periods.

Characterization of chikungunya virus induced host response in a mouse model of viral myositis.

While a number of studies have documented the persistent presence of chikungunya virus (CHIKV) in muscle tissue with primary fibroblast as the preferable cell target, little is known regarding the alterations that take place in muscle tissue in response to CHIKV infection. Hence, in the present study a permissive mouse model of CHIKV infection was established and characterized in order to understand the pathophysiology of the disease. The two dimensional electrophoresis of muscle proteome performed for differential analysis indicated a drastic reprogramming of the proteins from various classes like stress, inflammation, cytoskeletal, energy and lipid metabolism. The roles of the affected proteins were explained in relation to virus induced myopathy which was further supported by the histopathological and behavioural experiments proving the lack of hind limb coordination and other loco-motor abnormalities in the infected mice. Also, the level of various pro-inflammatory mediators like IL-6, MCP-1, Rantes and TNF-α was significantly elevated in muscles of infected mice. Altogether this comprehensive study of characterizing CHIKV induced mouse myopathy provides many potential targets for further evaluation and biomarker study.

Differential proteome analysis of Chikungunya virus-infected new-born mice tissues reveal implication of stress, inflammatory and apoptotic pathways in disease pathogenesis.

Chikungunya infection is a major disease of public health concern. The recurrent outbreaks of this viral disease and its progressive evolution demands a potential strategy to understand major aspects of its pathogenesis. Unlike other alphaviruses, Chikungunya virus (CHIKV) pathogenesis is poorly understood. In every consecutive outbreak, some new symptoms associated with virulence and disease manifestations are being reported such as neurological implication, increased severity and enhanced vector competence. In order to unravel the mechanism of the disease process, proteomic analysis was performed to evaluate the host response in CHIKV-infected mice tissues. Comparative analysis of the multiple gels representing the particular tissue extract from mock and CHIKV-infected tissues revealed a drastic reprogramming of physiological conditions through 35 and 15 differentially expressed proteins belonging to different classes such as stress, inflammation, apoptosis, urea cycle, energy metabolism, etc. from liver and brain, respectively. Based on the alterations obtained in the CHIKV mouse model, most of the aspects of CHIKV infection such as disease severity, neurological complications, disease susceptibility and immunocompetence could be defined. This is the first report unravelling the complicated pathways involved in the mechanism of Chikungunya disease pathogenesis employing proteomic approach.

Development and evaluation of antigen capture ELISA for early clinical diagnosis of chikungunya.

The resurgence of chikungunya (CHIK) in the form of unprecedented explosive epidemic after a gap of 3 decades in India and Indian Ocean islands is a point of major public health concern. The laboratory diagnosis is essentially based on virus isolation, IgM ELISA, and reverse transcriptase polymerase chain reaction (RT-PCR). Although PCR-based methods are used for early and accurate diagnosis, the high cost of the assay and requirement of thermal cycler limit its application only to referral laboratories. The antibody-based IgM ELISA is found to be cost-effective, but it takes 5 to 6 days for the patient to develop antibody and, thus, has less implication for early clinical diagnosis and patient management. Therefore, a simple rapid, sensitive, and specific antigen detection system is reported for early and reliable clinical diagnosis as well as effective surveillance of CHIK. A double antibody sandwich system was designed for antigen capture ELISA, employing rabbit and mouse anti-CHIK IgG antibodies as capture and detector antibodies, respectively. An optimal assay condition with 0 background was established having no reactivity with healthy human serum and Cerebro spinal fluid (CSF) samples. The comparative evaluation with SYBR Green I-based real-time RT-PCR revealed an accordance of 96% with a sensitivity and specificity of 95% and 97%, respectively. The specificity of this assay was confirmed through cross-reactivity studies with confirmed dengue and Japanese encephalitis (JE) patient serum and CSF samples. The antigen capture ELISA reported in this study was able to detect the presence of viral antigen as early as the second day of fever and, thus, can be very useful for early clinical diagnosis of CHIK with acute phase patient serum and CSF samples. This can also be used for rapid screening of large numbers of clinical samples in endemic areas during epidemics.