Potential Role For The Nonstructural Protein NS1 In Early Diagnosis And Serotyping Of Dengue Virus Infections

Dengue is one of the most important mosquito-borne viral diseases in humans predominantly affecting tropical and subtropical areas. The global prevalence of dengue has grown dramatically in recent decades. The disease is now endemic in more than 100 countries around the world and there may be 50 million to 100 million cases of dengue virus infections each year estimated by the World Health Organization. Small epidemics of dengue also frequently occurred in southern regions of our country, for example only in 2002 nearly 10 thousand people were affected in Guangdong and Macao. Dengue virus has four distinct serotypes (DV1, 2, 3, and 4). Infection with any of the four serotypes of dengue virus causes a spectrum of clinical features, ranging from asymptomatic infections, undifferentiated fever, and classical dengue fever (DF) to life-threatening manifestations, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Infection induces a life-long protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection by the other three serotypes. Seroepidemiological studies have shown that subsequent heterologous infection may be a major risk factor for DHF/DSS due to antibody-dependent enhancement. At present, however, there is no protective vaccine or specific treatment available for dengue virus infection. Thus, early clinical management can reduce the morbidity and mortality of DHF or DSS. Since symptoms of DV infections are insufficiently specific for accurate clinical differentiation from other febrile illnesses and hemorrhagic fever, the definitive diagnosis of DV infections relies on laboratory tests. A rapid and accurate dengue diagnosis in the acute phase of illness is important for enrolling patients in clinical trials for novel antiviral treatment or early enhancement of epidemiological control measures in areas with low endemicity. Furthermore, for epidemiological and pathological investigations, it is important to determine the serotypes of dengue virus which have different correlation with disease severity. Currently, laboratory diagnosis of DV infections is based on viral isolation, serology, and RNA detection. Viral isolation is a gold standard for the diagnosis and the serotype of DV infections, but this method is time-consuming and requires a sophisticated laboratory. Viral nucleic acid detection typically provides more sensitive and rapid diagnosis than traditional virus isolation method does. However, the molecular diagnosis requires experienced technicians and specialized laboratory equipment and ease to bring out false-positive results due to contamination. Although the detection of antibodies using whole virus antigens based enzyme-linked immunosorbent assay (ELISA) is the most commonly used, the limitations of this assay are cross-reactivity with all serotypes of DV and also other members of the flavivirus family. Therefore, early diagnosis and determination of serotype still remains a problem, as it mainly depends on RT-PCR or virus isolation methods.As an alternative, the detection of viral antigens has been proposed and more recently attention has been focused on nonstructural protein-1 (NS1) of DV. Dengue virus is a positive-stranded encapsulated RNA virus. The genomic RNA is composed of three structural protein genes that encode the nucleocapsid or core protein (C), a membrane-associated protein (M), an envelope protein (E), and seven nonstructural protein genes(NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). NS1 has been identified as a highly conserved glycoprotein expressed in either membrane associated or secreted forms. It possesses not only group-specific but type-specific determinants and has been recognized as an important immunogen in DV infections. Therefore NS1 protein might be as an early diagnostic marker and a serotyping marker. In this study, considering the fact that the mainly epidemic serotype in Guangdong during recent years is DV1, we developed and characterized a panel of monoclonal antibodies (mAbs) to serotype-specific epitopes of NS1 from DV1, by which a rapid and sensitive serotype-specific assay for the early diagnosis of DV infections was established. This two-site sandwich antigen-capture ELISA with highly affinity and highly specificity monoclonal antibodies was tested and optimized for detection of DV1NS1. The clinical application of this technology in the early diagnosis of DV infections and identification of the serotype of DV1 are further investigated.Ⅰ. Cloning and expression of DV1 nonstructural protein NS1 and identification of its antigenicityThe cDNA of nonstructural protein NS1 was amplified with RT-PCR from the C6/36 cells infected with DV1 and was cloned into the multi-cloning sites of pQE30 and expressed with induction of IPTG. After the purification under denature condition, the immunogenicity of the recombinant protein was identified by the rabbit sera against DV1 and acute sera of patients with DV1-infection through Western Blot and ELISA methods. Experimental results showed the recombinant NS1 protein was highly expressed in E. coli M15 and the purified NS1 could be recognized and combined by the serum against the dengue virus serotype 1 from the rabbit and patients infected with the virus. The expressed NS1 protein has good antigenicity, which may provide a potential source for studying the function of NS1 and use as an antigen for dengue serum detection.Ⅱ. Preparation and characterization of monoclonal antibodies against DV1 nonstructural protein 19 Balb/c mice were divided into three groups: one immunized by recombinant DV1NS1 protein which of high antigenicity, one immunized by inactive DV1, and one received several boost of recombinant DV1NS1 after two injections of inactive DV1. Then the splenocytes of the immunized mice were fused with myeloma cells to produce hybridoma cell line, which could secrete anti-DV1NS1 protein antibodies. Enzyme-linked immunosorbent assay (ELISA), immnofluorescence assay (IFA) and Western blot analysis were applied to identify specificity of antibodies. Ultimately 10 strains of hybridoma cell lines steadily secrete antibodies of nonstructual protein 1 were obtained. These antibodies had characteristics of specific binding to DV and recombinant NS1 protein. Among them, one strain was identified as IgG2a isotype and the other nine were all IgG1. Nine out of the ten strains were specific to DV1 without detectable cross-reactivity with the other three serotypes of dengue virus. Competitive inhibition assay showed that these mAbs recognized at least six distinct epitopes of NS1 antigen. These obtained antibodies with high activity and specificity will provide a potential value for vaccine researches and early diagnosis of dengue virus infection.Ⅲ. Establishment of the antigen capture enzyme-linked immunosorbent assay for detection dengue virus serotype 1 nonstructural protein NS1To develop the mAb-based antigen capture enzyme-linked immunosorbent assay (ELISA) for DV1NS1, the antibodies were paired one by one based on their recognizing epitopes, binding affinity and antibody titers to choose the optimal coating mAb and detecting mAb through determination the detection sensitivity and specificity. The best coating antibody and HRP-conjugate detection antibody selected ultimately to construct the antigen capture ELISA was 1F31A5 and HRP-5141A3. The lowest limit of detection of the recombinant NS1 protein with this capture ELISA is 5ng/ml approximately and the detection linear range is approximately 100～2000ng/ ml. Furthermore, this mAb-based antigen-capture assay is specific to DV1NS1 and has no cross-reactivity with either other serotypes of DV or other closely related members of the flavivirus family (Japanese encephalitis and Yellow fever) when the virus-infected cell culture was used. All these results demonstrated that the established test is sensitive and specific which may lay the basis for providing a novel laboratory diagnosis tool for dengue virus infection.Ⅳ. The preliminary application study of the antigen capture enzyme-linked immunosorbent assay for detection DV1 nonstructural protein NS1 in sera specimensIn this part the developed antigen-capture ELISA was evaluated for its early detection and serotyping of dengue virus in sera samples. To establish the baseline of the normal range in DV1NS1 antigen-capture assay for clinical evaluation, serum specimens from 469 healthy blood donors were analyzed. Only5 of 469 sera from healthy blood donors were defined as low level false-positive with the detection specificity of 99%. A total of 462 serum specimens from clinical probable DV1-infected patients during the DV1 epidemic in Guangdong in 2002-2003 were analyzed. The DV1NS1 was detectable in blood circulation from the first day up to day 18 after onset of symptoms. The percentage of DV1NS1 positive samples was 52.8% on day 1 to 2, peaked at 83.8% on day 6 to 10, and decreased to 50% on day 11 to 15 after the onset of symptoms. Using the same panel of serum specimens, IgM antibodies against dengue virus were also measured by MAC-ELISA, which is currently commercially available for routine dengue diagnosis. The positive rate of anti-DV IgM was only 17.4% on day 1 to 2, and increased from 75% during 6-10 days to 80% during 16-20 days after onset. Obviously, NS1 and IgM were detected concomitantly during the acute phase, but at earlier times especially from day 1 toward day 3, the NS1 protein showed a more sensitive detection. The sensitivity of DV1NS1 detection in sera with reference to the results of RT-PCR was 82%. All these results demonstrated that NS1 may be more suitable for the early detection of DV infections.The assay for serotype-specific NS1 of DV1 was further demonstrated in clinical serum samples. A limited number of acute-phase serum specimens from 20 DV2-infected patients and one DV3-infected patient confirmed by both virus isolation assay and RT-PCR were analyzed. None of these serum specimens were detected in the DV1NS1 antigen-capture ELISA, indicated that the antigen-capture ELISA is highly specific for DV1NS1 detection. In addition, DV1NS1 test results with 13 serum specimens from patients with JE virus infections, 51 specimens from patients with Hantan virus infections, 56 specimens from patients with measles, and 20 specimens from patients with leptospirosis were all negative, indicating that the assay is highly specific for the DV1 with no cross-reactivity with either other flaviviruses or nonflavivuses infections. These findings suggested that the serotype-specific mAb-based NS1 antigen-capture ELISA might be a valuable tool for early diagnosis and serotyping of DV infections.In conclusion,1. The DV1NS1 protein was expressed and purified successfully which has good antigenicity, and a panel of mAbs specific to the NS1 of DV1 was successfully obtained. This may provide a potential source for studying the function of NS1 and for vaccine researches and early diagnosis of dengue virus infections.2. A mAb-based antigen-capture ELISA for detection circulating NS1 protein was successfully established. The high detection sensitivity makes it suitable for the early detection of DV infections. This method may also be developed as a quantitative reagent to provide disease severity stratification.3. A DV1-specific mAb-based antigen-capture ELISA has been developed. It has high detection specificity to DV1 without cross-reactions with either other three DV serotypes or other closely related members of the flavivirus. The characteristic will be helpful in rapid serotyping and differential diagnosis of dengue virus infections.