Novel Immunofluorescence Assay Using Recombinant Nucleocapsid-Spike Fusion Protein as Antigen To Detect Antibodies against Severe Acute Respiratory Syndrome Coronavirus

Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a “gold standard” during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.     

Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay and Immunochromatographic Tests for Detection of Immunoglobulin G Antibodies to Severe Acute Respiratory Syndrome (SARS) Coronavirus in SARS Patients

An enzyme-linked immunosorbent assay (ELISA) and a rapid immunochromatographic test for detection of immunoglobulin G (IgG) antibodies in severe acute respiratory syndrome (SARS) patients were developed by utilizing the well-characterized recombinant proteins Gst-N and Gst-U274. The ELISA detected IgG antibodies to SARS-CoV in all 74 convalescent-phase samples from SARS patients while weakly cross-reacting to only 1 of the 210 control sera from healthy donors. This finding thus led to a kit sensitivity, specificity, and accuracy of 100, 99.5, and 99.6%, respectively. The test thus provided a positive predictive value (PPV) of 98.7% and a negative predictive value (NPV) of 100%. In addition, the ELISA gave a positive delta of 5.4 and a negative delta of 3.6, indicating an excellent differentiation between positives and negatives. The same recombinant proteins were also applied to a newly developed platform for the development of a 15-min rapid test. The resulting rapid test has an excellent agreement of 99.6%, with a kappa value of 1.00, with the ELISA. Again, this rapid test was able to detect 100% of the samples tested (n = 42) while maintaining a specificity of 99.0% (n = 210). The PPV and NPV for the rapid test thus reached 95.3 and 100%, respectively.     

Retrospective Serological Investigation of Severe Acute Respiratory Syndrome Coronavirus Antibodies in Recruits from Mainland China

Different assays were used to analyze 1,621 serum specimens collected from military recruits from the People's Republic of China in 2002 for severe acute respiratory syndrome (SARS) coronavirus antibodies. The results demonstrated that the subjects either had rarely been exposed to the virus before the 2003 SARS outbreak or had not been exposed but the nucleocapsid protein cross-reacted with other antibodies in humans.     

Validation of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most significant diseases of swine. IDEXX HerdChek PRRS, a commercially available enzyme-linked immunosorbent assay (ELISA), has become the industry standard for the detection of antibodies against PRRS virus (PRRSV). The need to accurately determine the PRRSV serostatus of herds and individual animals has prompted the development of several follow-up assay methods. A highly specific and repeatable blocking ELISA (bELISA) was developed on the basis of the use of an expressed PRRSV nucleocapsid (N) protein as the antigen and a biotinylated monoclonal antibody. Validation of the bELISA used sera from 316 animals experimentally and naturally infected with North American PRRSV and sera from 370 uninfected animals. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 97.8% and a diagnostic specificity of 100%. The between-run coefficient of variation of an internal quality control serum was 4.24%. The bELISA was able to detect seroconversion as well as the IDEXX ELISA and the indirect fluorescent antibody (IFA) assay; kappa values were 0.94 and 0.96, respectively. A collection of 133 serum samples with unexpected positive IDEXX ELISA results was obtained from 4,038 diagnostic samples submitted from farms from which PRRS-negative results were expected. The bELISA identified 97% of the samples as PRRS seronegative, while the IFA identified 100% as seronegative. The anticipated use of the bELISA is as a follow-up test to the IDEXX ELISA for determining the PRRSV serostatus of individual animals with unexpected positive test results from swine herds from which negative results are expected.     

Evaluation of Two Immunoblot Assays and a Western Blot Assay for the Detection of Antisyphilis Immunoglobulin G Antibodies

In the present study, two immunoglobulin G (IgG) immunoblot assays and one IgG Western blot assay were compared to the rapid plasma reagin test (RPR), the fluorescent treponemal antibody absorption test (FTA-ABS), and the Treponema pallidum particle agglutination assay (TP-PA). The agreement levels of the Viramed, Virotech, and MarDx assays were 97.0%, 96.4%, and 99.4%, and the agreements of samples inconclusive by FTA-ABS and resolved by TP-PA were 91.7%, 83.3%, and 69.4%, respectively.Syphilis, a disease caused by Treponema pallidum, is transmitted congenitally or through sexual intercourse (8-9). Non-treponema-based tests such as the rapid plasma reagin test (RPR) are used to detect syphilis infection (6, 9-10). These tests may produce false-positive results in pregnant women and patients with infections (3, 5-6, 9, 11). An algorithm has been developed for the serological diagnosis of syphilis which includes a non-treponema-based screening test and a treponema-based confirmatory assay (1-2, 7, 11). Traditional confirmatory assays include the fluorescent treponemal antibody absorption test (FTA-ABS) and the T. pallidum particle agglutination assay (TP-PA) (9).Western blot-based assays to detect immunoglobulin G (IgG) antibodies may prove useful, especially in cases where the FTA-ABS is inconclusive. In the present study, results of two immunoblot assays and one Western blot assay were compared to FTA-ABS/TP-PA and RPR results, as well as to each other.     

Dot Blot Assay for Detection of Antidiacyltrehalose Antibodies in Tuberculous Patients

A simple dot blot test with diacyltrehalose (DAT) as the antigen was developed to detect anti-DAT antibodies in tuberculous patients. To enhance antigen-antibody reaction detection, rabbit serum raised against human immunoglobulins was used prior to incubation with a protein A-colloidal gold complex. With the dot blot system, it was possible to obtain a sensitivity similar to that of enzyme-linked immunosorbent assay (ELISA) and a specificity of 97.14%, versus a specificity of 94.29% by the ELISA. We conclude that this simple and fast assay could be used in places where ELISA equipment is not easy available and that it might also be applicable with other Mycobacterium tuberculosis immunogenic antigens.     

Development of a Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Quantification of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus

A rapid, inexpensive enzyme-linked immunosorbent assay (ELISA) to quantitate antibodies to porcine respiratory and reproductive syndrome virus (PRRSV) in serum was developed using a recombinant PRRSV nucleoprotein (rN). The sensitivity (85.3%) and specificity (81.7%) of the Kansas State University ELISA were good, correlating well (82.4%) with the IDEXX HerdChek ELISA.     

Use of the COOH Portion of the Nucleocapsid Protein in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay for Specific and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus

Antibody detection with a recombinant COOH portion of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (N) protein, N13 (amino acids 221 to 422), was demonstrated to be more specific and sensitive than that with the full-length N protein, and an N13-based antigen-capturing enzyme-linked immunosorbent assay providing a convenient and specific test for serodiagnosis and epidemiological study of SARS was developed.     

Evidence of increasing diversification of emerging Severe Acute Respiratory Syndrome Coronavirus 2 strains

On 30th January 2020, an outbreak of atypical pneumonia caused by a novel betacoronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a public health emergency of international concern by the World Health Organization. For this reason, a detailed evolutionary analysis of SARS-CoV-2 strains currently circulating in different geographic regions of the world was performed. A compositional analysis as well as a Bayesian coalescent analysis of complete genome sequences of SARS-CoV-2 strains recently isolated in Europe, North America, South America, and Asia was performed. The results of these studies revealed a diversification of SARS-CoV-2 strains in three different genetic clades. Co-circulation of different clades in different countries, as well as different genetic lineages within different clades were observed. The time of the most recent common ancestor was established to be around 1st November 2019. A mean rate of evolution of 6.57 × 10⁻⁴ substitutions per site per year was found. A significant migration rate per genetic lineage per year from Europe to South America was also observed. The results of these studies revealed an increasing diversification of SARS-CoV-2 strains. High evolutionary rates and fast population growth characterizes the population dynamics of SARS-CoV-2 strains.     

Profiles of Antibody Responses against Severe Acute Respiratory Syndrome Coronavirus Recombinant Proteins and Their Potential Use as Diagnostic Markers

A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.     

Genomic Evolution of Severe Acute Respiratory Syndrome Coronavirus 2 in India and Vaccine Impact

Recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and subsequent containment procedures have impacted the world as never seen before. Therefore, there is considerable curiosity about the genome evolution related to the origin, transmission and vaccine impact of this virus. We have analysed genome sequences of SARS-CoV-2 isolated from Indian patients to gain an in-depth understanding of genomic evolution and transmission in India. Phylogenetic analysis and mutation profiling revealed major lineages being evolved by characteristic mutations. As the mutation frequency in spike protein is comparatively lesser, the candidate vaccines expected to have wide coverage worldwide including India.     

Profile of Antibodies to the Nucleocapsid Protein of the Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus in Probable SARS Patients

Profiles of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)-associated coronavirus in 445 probable SARS patients and 3,749 healthy people or non-SARS patients were analyzed by antigen-capturing enzyme-linked immunosorbent assay. Antinucleocapsid antibodies were elucidated in 17.5% of the probable SARS patients 1 to 7 days after the onset of symptoms and in 80% of the patients 8 to 14 days after the onset. About 90% of the probable SARS patients were positive 15 or more days after illness. Antibody titers increased up to 70 days, and high antibody titers were maintained at least for another 3 months. Of the healthy people and non-SARS patients, only seven (0.187%) were weakly positive.     

Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection and Differentiation of Antibodies against European and North American Porcine Reproductive and Respiratory Syndrome Virus

Two types of porcine reproductive and respiratory syndrome virus (PRRSV) have been reported, the European type (EU PRRSV) and the North American type (US PRRSV). We developed a dual enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection and differentiation of serum antibodies directed against either of the two PRRSV types. This tandem PRRS ELISA is based on affinity-purified recombinant nucleocapsid protein expressed in Escherichia coli. Sensitivity and specificity were assessed by using the IDEXX HerdChek PRRS ELISA and the indirect immunofluorescence assay as reference tests. A total of 1,571 sera originating from the United States, Europe, and two PRRS-free countries, i.e., Switzerland and New Zealand, were used for validation of the tandem PRRS ELISA. The new test performed at least as well as the reference tests in regard to sensitivity (0.94 for the US PRRS ELISA and 0.93 for the EU PRRS ELISA) and specificity (0.96 for the US PRRS ELISA and 0.99 for the EU PRRS ELISA). Positive sera were correctly differentiated in 582 of 591 cases, indicating a high differentiation capability of this dual ELISA. The robustness and repeatability of the test were assessed and found to be appropriate for diagnostic applications. Taken together, the data indicate that the tandem PRRS ELISA described here is the first differentiation ELISA for PRRSV serology based on recombinant antigen. It is convenient with respect to antigen production, and it is reliable, economical, and highly sensitive and specific. Thus, it is considered to be a powerful tool for routine diagnostics, epidemiological surveys, and outbreak investigations.