Development Of Immunological Methods For Detection Of Chikungunya Virus Infection

Chikungunya virus(CHIKV)is a single positive strand RNA virus,which belongs to the Togaviridae family,alphavirus genus.The virus particles are icosahedral T = 4 symmetry with diameters of 60-70 M,.The virus has five structural proteins(C,E2,E3,6K,E1),E1 and E2 form heterodimers,and three E1/E2 heterodimers to form a tiny spike.The surface of each virus particles has a total of 80 identical spikes.The E1/E2 heterodimer is attached to the capsid of the C protein under the envelope.In addition to structural proteins,there are four non-structural proteins(NS 1,NS2,NS3,NS4)mainly play a role in the regulation of viral replication process.People infected with CHIKV develops Chikungunya fever,a self-limiting disease,most patients have mild symptoms,such as fever,rash,headache.A small portion of patients will be complicated by encephalitis and other serious symptoms.CHIKV spread through Aedes mosquitos that have a wide range of distribution in the world.The Southeast Asian region,which is close to China,has been one of the main endemic areas of Chikungunya.In China,CHIKV first isolated in 1986,in 2008,China's first imported Chikungunya fever case was reported.In 2010 Chikungunya fever outbreak in Dongguan,caused by imported cases.Early diagnosis is very important for prevention and control of infectious disease.We established rapid CHIKV antigen and antibodies detection methods based on baculovirus expressed.CHIKV virus-like particle(VLP),which contains all the structural proteins of CHIKV.The antigenicity of VLP is similar to inactivated CHIKV.Because VLP does not contain viral nucleic acids and can be operated without biosafety risk,greatly reducing the limitations of the study.Mice ascites and rabbit serum containing anti-CHIKV polyclonal antibody were produced by using VLP immunized mice and rabbits.The antibody titers were more than 1:10 00000 by ELISA.1.A double antibody sandwich ELISA for the detection of CHIKV antigens was established by purified rat ascites and rabbit serum.The specificity and detection limit of double antibody sandwich ELISA was satistying,50TCIDso virus can be detected by using mimic patients serum and the serum of dengue fever,hemorrhagic fever with renal syndrome patients',and healthy human were negative.The variation between the plate was less than 10%,and that of intraplate variation less than 5%.2.VLP based indirect ELISA and MacELISA for the detection of anti-CHIKV IgG and anti-CHIKV IgM were established.The antigenicity of VLP is equal to inactivated virus as antigen for antibody detection.IgG and IgM antibodies to CHIKV were successful ditected in patients' sera by using VLP based indirect ELISA and MacELISA.The variation between the plate less than 10%,intraplate variation less than 5%.In this study,developed and preliminary evaluated the assay methods of CHIKV antigen and antibodies,and it will play a great role for clinical diagnosis and epidemiological investigation.