Chikungunya fever (CHIKF), a mosquito-borne infectious disease with fever, rash, muscle pain and arthralgia as the main symptoms caused by chikungunya virus (CHIKV) infection, was first detected in southern Tanzania in 1952. CHIKV is an RNA virus belonging to the genus Alphavirus of the family Phiviridae, which usually circulates in a curved cycle between non-human primates, mammalian hosts, and mosquitoes. Given the frequent movement of mosquitoes in endemic and non-endemic areas of the world, native Chinese mosquitoes may also already have the ability to transmit the virus. Therefore, it is urgent to improve the level of awareness and surveillance of this disease on the mainland.
Figure 1 Life cycle of chikungunya virus: interrelated between forest and urban cycles
CHIKV is highly contagious and is considered one of the fastest spreading mosquito-borne infectious diseases after malaria and dengue fever. There is currently no vaccine or specific drug against the virus. Because its clinical signs and symptoms are similar to dengue fever and Zika, it is very easy to be misdiagnosed in these common areas, and once a cluster of outbreaks occurs, it will have a serious spillover effect. Therefore, it is crucial for the differential diagnosis of this disease. Methods used to diagnose CHIKV infection include virus isolation, viral genome assays, or enzyme-linked immunoassays (ELISA). Fluorescence quantitative PCR (qRT-PCR) analysis is a viable alternative to viral nucleic acid detection compared to cell culture. However, the experimental conditions of this technique are demanding, time-consuming and the rate of missed detection is extremely high. Antibody-based ELISA serological testing is a simple, effective method for identifying and quantifying viral antigens. According to the survey, the commercial ELISA is mainly imported, expensive, and is only used to detect IgM and IgG antibodies in CHIKV patients. Sustained global trade and growth in international travel may increase the probability of mosquito-borne viruses, and there is a need to screen feverish entrants for CHIKV infection using rapid diagnostic reagents, such as at airports or ports of entry and exit, to reduce the risk of import and transmission within the country.
Recently, Professor Lu Jiahai's team from the School of Public Health of Sun Yat-sen University published a research paper entitled Highly potent multivalent VHH antibodies against Chikungunya isolated from an alpaca naïve phage display library at the Journal of Nanobiotechnology. For the currently immature detection of CHIKV E2 antigen ELISA finished products, the use of alpaca source natural bacteriophage library, with CHIKV E2 antigen as the target, through the biological selection method to screen nano-antibodies, based on this antibody to establish a convenient and fast ELISA serological detection method.
Figure 2 Protocol
Compared with traditional antibodies, nano-antibodies have the advantages of structural stability, high affinity, not easy to aggregate, can recognize potential antigen epitopes, and are easy to humanize, and are often used as research tools such as structural biology, cell biology and developmental biology. Through three rounds of biological selection, 20 specific nano-antibodies were successfully screened. Among them, the nano-antibodies Nb-2E8 and Nb-3C5 were modified in tandem, and it was found that they bound to the specific high affinity of E2 protein in the nanomolal range. The results showed that the polyvalent nano-antibodies Nb-2E8 and Nb-3C5 showed high affinity compared with monovalent antibodies. The increased affinity by more than 100 times compared to the monovalent form indicates that it can be applied to detect CHIKV virus particles in serum and help with disease diagnosis, drug therapy, and vaccine development.
Figure 3 Kinetic analysis of the interaction between chikv E2 protein and nanoantibodies
Using BiNb-2E8 as the capture antibody and HRP-bound BiNb-3C5 as the detection antibody, a nano-antibody sandwich method ELISA was established. In the range of 5-1000 ng/mL, a good linear relationship (r=0.9864, P<0.0001) was obtained between OD450 values and E2 protein concentrations, indicating that it has the potential for quantitative detection of E2 proteins.
Figure 4 A multivalent nano-antibody chikungunya virus sandwich ELISA detection method
The above results show that the nano-antibodies screened in this study can be applied to the preparation of rapid and convenient CHIKV detection reagents, providing strong technical support and raw material support for the rapid and accurate diagnosis of CHIKV. This study is based on the multidisciplinary cross-integration of the actual needs of mosquito-borne infectious diseases and major scientific issues, which helps to improve the diagnostic level of mosquito-borne infectious diseases and achieve collaborative innovation. The core of One Health is to solve the problems of monitoring, early warning and response to emerging infectious diseases through interdisciplinary, cross-departmental and cross-regional cooperation, and improve the survival and quality of life of people and animals. In the future, we will learn from the same health concept and further deepen cooperation and exploration in the field of mosquito-borne infectious disease vaccines. It also looks forward to multidisciplinary, cross-sectoral and cross-integrated approaches to focusing on human and animal health and addressing many challenges.
Li Qianxuan, a doctoral student at the School of Public Health of Sun Yat-sen University, is the first author of the paper, Professor Lu Jiahai is the corresponding author of the paper, and the School of Public Health of Sun Yat-sen University is the first unit to complete the work.
This article is reprinted from the Sun Yat-sen University News Network
Original link:
https://doi.org/10.1186/s12951-022-01417-6
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